Tree Genetics & Genomes (2012) 8:709–723
DOI 10.1007/s11295-011-0458-3

ORIGINAL PAPER

Aligned genetic linkage maps of apple rootstock cultivar‘JM7’ and Malus sieboldii ‘Sanashi 63’ constructedwith novel EST-SSRs
Shigeki Moriya & Hiroshi Iwanami & Nobuhiro Kotoda &Takashi Haji & Kazuma Okada & Shingo Terakami &Naozumi Mimida & Toshiya Yamamoto & Kazuyuki Abe

Received: 27 May 2011 / Revised: 8 November 2011 / Accepted: 21 November 2011 / Published online: 30 December 2011
# Springer-Verlag 2011

Abstract Identification of markers associated with genes ofinterest and quantitative trait loci (QTLs), combined withhigh-density genetic linkage maps, can help reduce laborand costs by enabling marker-assisted selection (MAS). In
this study, a dwarfing apple rootstock cultivar ‘JM7’ (Malus
prunifolia × Malus pumila ‘Malling 9’) and wild apple
Malus sieboldii ‘Sanashi 63’ (section Sorbomalus) wereused for constructing genetic linkage maps. Here, a species
from section Sorbomalus was used for the first time as atarget species in a genome-wide mapping study. We also

Communicated by E. Dirlewanger
Electronic supplementary material The online version of this article(doi:10.1007/s11295-011-0458-3) contains supplementary material,
which is available to authorized users.
S. Moriya (*) : H. Iwanami : N. Kotoda : T. Haji : K. Okada :N. Mimida : K. AbeApple Research Station, NARO Institute of Fruit Tree Science,
Shimokuriyagawa,Morioka, Iwate 020-0123, Japane-mail: moriyas@affrc.go.jp
S. Terakami : T. Yamamoto
NARO Institute of Fruit Tree Science,
Fujimoto,
Tsukuba, Ibaraki 305-8605, Japan
Present Address:
N. Kotoda
Okitsu Citrus Research Station,
NARO Institute of Fruit Tree Science,
Shimizuokitsunaka-cho,
Shizuoka, Shizuoka 424-0292, Japan
Present Address:
N. Mimida
Faculty of Agriculture, Iwate University,
Morioka, Iwate 020-8550, Japandeveloped and mapped 137 novel-expressed sequence tagsimple sequence repeat (EST-SSR) markers. The genetic
linkage maps of ‘JM7’ and ‘Sanashi 63’ consisted of 415
and 310 loci and spanned 998.0 and 981.8 cM, respectively,
comparable to the reference map of Malus × domestica‘Discovery’. A BLASTN search revealed that all of the
EST-SSR sequences used in this study exhibited very highhomology to one or more previously characterized apple
genome contigs. Although the most homologous contigs
of 89 EST-SSRs were located within the same linkage
groups (LGs) identified by mapping analysis, the other 48
EST-SSRs were aligned into contigs positioned in different
LGs than those identified by mapping. When search criteria
were expanded to include the five most homologous contigs
of each EST-SSR, at least one of the top five contigs for 15of these 48 EST-SSRs corresponded to the LG obtained by
mapping. The maps of ‘JM7’ and ‘Sanashi 63’ may be
useful for analyzing important rootstock characteristics andidentifying markers for MAS.
Keywords EST-SSR . Genetic linkage map . Malus
sieboldii . Rootstock

Introduction
Apple (Malus × domestica Borkh.) is a major fruit tree intemperate regions, producing 70 million tons of fruit in 2008
according to the Food and Agriculture Organization of theUnited Nations (http://faostat.fao.org/faostat/). Apple cultivation management methods such as planting density andpruning methods depend on the rootstocks used. Excellent
rootstocks derived from Malus spp. are essential for commercial apple cultivation because it is necessary to graft

710

fruiting scions onto rootstocks. Apple rootstock breeding
has been conducted worldwide, resulting in the development rootstock cultivars such as the “M” series from theEast Malling Research Station, UK (Hatton 1917), the “G”
series from Cornell University, USA (Robinson et al. 2003),
and the “P” series by Skierniewice, Poland (Zagaja 1980).
The National Institute of Fruit Tree Science (NIFTS, Morioka, Japan) developed the “JM” series rootstocks, whichchange the tree growth habit to dwarf or semi-dwarf andwere easy to propagate from hardwood cuttings (Soejima etal. 2010). Since crown gall disease recently became a problem in nurseries, rootstock breeding programs began to
introduce genes conferring resistance to crown gall disease
into “JM” rootstocks (Moriya et al. 2008). However, evaluations of vigor, ease of propagation, and crown gall resistance require time- and labor-intensive processes forhandling many individuals and repeated trait assessmentsby traditional breeding methods. Identification of markers
associated with genes of interest and quantitative trait loci
(QTLs), combined with high-density genetic linkage maps,
can help to reduce labor and costs by enabling markerassisted selection (MAS). However, not enough simple sequence repeat (SSR) markers, especially expressed sequence tag (EST)-derived SSR markers, have beendeveloped to allow marker-assisted breeding in apple rootstocks, and very few functional genes have been employed
as selection markers.
Despite the broad range of apple species used for rootstocks (Luby 2003), genomics approaches to rootstock
breeding have been limited. The genus Malus is divided
into five sections (Malus, Sorbomalus, Eriolobus, Choromeles, and Docyniopsis) based on morphological traits andflavonoid similarities (Phipps et al. 1990). Among these,
sections Malus and Sorbomalus have been utilized frequently as rootstocks. However, all of the previously reported
genome-wide genetic linkage maps have been constructedusing only species belonging to the section Malus which
includes the fruiting scion apple (M. × domestica).
Researchers have searched for molecular markers associatedwith genes of interest for rootstocks such as dwarfing(Pilcher et al. 2008), resistance to woolly apple aphid (Buset al. 2008), and crown gall disease resistance (Moriya et al.
2010). Dwarfing and resistance to woolly apple aphid werestudied using genetic linkage maps of F1 progeny obtainedby crossing ‘Malling 9’ (M.9; Malus pumila) with ‘Robusta
5’ (R.5; Malus baccata × Malus prunifolia) (Celton et al.
2009). Until now, these were the only genetic maps containing abundant informative markers that had been constructed
for rootstock. However, both mapping parents used by
Celton et al. (2009) belong to section Malus, so genetic
information from species outside of section Malus has been
insufficient. The only genetic linkage maps that have beenreported for species outside of section Malus were two

Tree Genetics & Genomes (2012) 8:709–723

partial maps constructed using Malus sieboldii, which
belongs to section Sorbomalus. One was for linkage group(LG) 8 of ‘Aotea 1’ (Bus et al. 2008), and the other was LG
2 of ‘Sanashi 63’ (Moriya et al. 2010). The LG 2 map of‘Sanashi 63’ was used to map the crown gall disease resistance gene Cg. Because it has been suggested that both the
major gene Cg and several QTLs are involved in crown galldisease resistance (Moriya et al. 2010), integrated and highdensity genetic linkage maps will be required to conduct
more detailed mapping and perform marker-assisted selection (MAS) for resistance.
SSR markers, which can be derived from either genomic
or EST sequences, are the most efficient molecular markers
with which to construct the landmarks and frameworks ofgenetic linkage maps. Until now, 298 SSR markers fromapple genomic sequences have been developed (Guilford etal. 1997; Gianfranceschi et al. 1998; Hokanson et al. 1998;
Liebhard et al. 2002; Hemmat et al. 2003; SilfverbergDilworth et al. 2006; Fernandez-Fernandez et al. 2008;
Celton et al. 2009). Many SSR markers have exhibited
transferability across genera between Malus and Pyrus,
which could help to identify colinearity between Malusand Pyrus. In particular, EST-based SSRs are located aroundconserved coding regions, and they are often more transferable between species and genera than are genomic SSRs
(Decroocq et al. 2003). Several sequencing projects havegenerated more than 250 000 ESTs in apple (Newcomb et al.
2006; Naik et al. 2006; Gasic et al. 2009a). Although someof these sequences contain SSRs that have been developed
into EST-SSR markers and mapped on linkage maps
(Silfverberg-Dilworth et al. 2006; Celton et al. 2009; van
Dyk et al. 2010), the number of established EST-SSRs has
been limited to about 190.
In this study, we developed 137 novel EST-SSR markers
and constructed integrated high-density genetic linkage
maps of ‘JM7’ and ‘Sanashi 63’. These maps were compared with the reference maps of the domesticated apples
cultivars ‘Fiesta’ and ‘Discovery’, and their genome synteny
is discussed.

Materials and methods
Plant materials and genomic DNA extraction
An interspecific cross made in 1995 between rootstock
cultivar ‘JM7’ (section Malus) and M. sieboldii ‘Sanashi
63’ was used for genome mapping; the mapping population
used here consisted of 120 F1 plants. ‘JM7’ originated fromthe cross M. prunifolia ‘Morioka Seishi’ × M. pumila ‘M.9’.
The parents on rootstocks and F1 progeny on their own roots
were grown in the orchard of the Apple Research Station of
NIFTS and were cut back to 1-m height each dormant

Tree Genetics & Genomes (2012) 8:709–723

season. Young leaves of 11-year-old F1 plants and theparents were harvested in July and stored at −80°C until
use. Genomic DNA was isolated according to the method
described by Moriya et al. (2010).
Construction of cDNA libraries
Six cDNA libraries were constructed from flower buds,
young fruits, mature fruits, and leaves of ‘Fuji’ and fromfruit peels and shoot apices of ‘Jonathan’ (both Malus ×
domestica). Total RNA was extracted using a cetyltrimethylammonium bromide-based method, as described by Kotoda
et al. (2000). Messenger RNA was isolated from total RNA
using a PolyATract mRNA purification kit (Promega, Madison, WI, USA), and six cDNA libraries were constructedwith a Zap-cDNA Synthesis Kit (Agilent Technologies,
Santa Clara, CA, USA) and Packagene Lambda DNA Packaging System (Promega). After converting each cDNA
phage library into a plasmid library by in vivo excision,clones with a cDNA insert were randomly sequenced using
an ABI 3730xl automated sequencer (Life Technologies,
Carlsbad, CA, USA). A total of 17 774 EST sequences were
processed to remove the vector and unreliable sequencesusing Pregap4 software (Bonfield et al. 1995), and sequences longer than 200 bp were extracted for contig assembly
using the Gap4 software program (Bonfield et al. 1995).
After the redundant clones were removed, contig assembly of raw sequences produced 9010 non-redundant sequences. Homology searches within databases of Arabidopsis
proteins (The Arabidopsis Information Resource (TAIR);
http://www.arabidopsis.org/) and contigs from Apple v1.0
genome assembly (Velasco et al. 2010; the Genome Database for Rosaceae (GDR); http://www.rosaceae.org/) were
performed using BLASTX and BLASTN, respectively. We
used the criterion that E-values under 1E-9 indicated significant homology.
EST-SSR development
The 9010 non-redundant sequences were searched for SSR
motifs. Primer pairs were designed based on sequences
flanking SSR motifs, which were identified using a searchengine available at the GDR homepage. The minimum
number of SSR repeats for marker development was set asfive repeats for dinucleotide motifs, four repeats for trinucleotide motifs, and three repeats for tetranucleotide motifs.
All primer pairs were designed to obtain amplicons ranging
between 60 and 350 bp in length.
PCR amplification was performed in a 10-μl solution of
1× Ex Taq buffer (Takara, Otsu, Japan), 0.20 mM of dNTPs,
0.4 pmol of each forward primer containing 17 bp (5′AATACGACTCACTATAG-3′) of tail sequence, 2 pmol ofreverse primer containing 7 bp (5′-GTTTCTT-3′) of pigtail

711

sequence, 0.25 U of TaKaRa Ex Taq (Takara), and 10 ng ofgenomic DNA. Amplification was performed as follows:
initial denaturation at 94°C for 5 min, followed by ten cycles
at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.
Then, 2 pmol of T7 universal primer (5′-AATACGACTCACTATAG-3′), labeled with fluorescent chemical D2,
D3, or D4, was added to each amplification tube, and asecond amplification was performed for 30 cycles at 94°C
for 1 min, 52°C for 1 min, and 72°C for 1 min followed by afinal extension at 72°C for 7 min. Primer sequences for the
EST-SSRs mapped in this study are given in Table 1.
PCR products were separated and detected with a CEQ
8000 Genetic Analyzer (Beckman-Coulter, Inc., Brea, CA,
USA). PCR product sizes were determined by using a Size
Standard Kit-400 for the CEQ 8000 (Beckman-Coulter).
Analysis of selected landmark SSRs
A total of 275 previously reported SSR markers from twogenera of the Rosaceae, Malus and Pyrus were tested for theF1 population between ‘JM7’ and ‘Sanashi 63’. Of these,
256 SSRs were derived from Malus (Guilford et al. 1997;
Gianfranceschi et al. 1998; Liebhard et al. 2002; Hokanson
et al. 1998; Silfverberg-Dilworth et al. 2006; FernandezFernandez et al. 2008; Celton et al. 2009; Costa et al.
2008), and 19 SSRs were from Pyrus (Yamamoto et al.
2002a, b; Sawamura et al. 2004; Nishitani et al. 2009). Afterscreening for SSR markers showing polymorphism within
one or both of the parental cultivars, 166 SSR markers wereused for genotyping of the 120 F1 progeny individuals.
SSR-PCR was performed by the method described aboveor according to Moriya et al. (2011).
Amplified fragment length polymorphism analysis
Amplified fragment length polymorphism (AFLP) analysis
was performed using an AFLP Analysis System II kit (LifeTechnologies), according to the supplier's protocol, exceptthat we used EcoRI primers labeled with a fluorescentchemical instead of non-labeled primers. Genomic DNA(200 ng) was digested with restriction enzymes MseI andEcoRI; the digested DNA fragments were then ligated to theadapters provided in the kit. Pre-amplification reactions
were performed with the supplied pre-amp primer mix.
Selective amplification was performed with 16 primer combinations of five Mse primers (KeyGene codes: M48, M50,
M59, M61, and M62) and eight Eco primers (KeyGene
codes: E31, E32, E33, E34, E39, E40, E41, and E42).
Selective primers were synthesized at BEX (Tokyo, Japan).
The PCR products were separated and detected using a
PRISM 3100 DNA sequencer (Life Technologies). The sizeof each amplified band was determined using internalstandard DNA (HD400ROX, Life Technologies) and712

Tree Genetics & Genomes (2012) 8:709–723

Table 1 Characteristics of novel EST-SSRs mapped in linkage maps of ‘JM7’ (J) and/or ‘Sanashi 63’ (S)NameAccession

Forward primer
sequence

Reverse primer
sequence

Motif

Type ofmarker

Map position(linkage group)‘JM7’

MEST001
MEST003
MEST006
MEST007
MEST008
MEST009
MEST011
MEST012
MEST013
MEST014

AB627185
AB627186
AB627187
AB627188
AB627189
AB627190
AB627191
AB627192
AB627193
AB627194

agagagcactcttcccacca
ccccgttaacccctcataat
gcattctccgacaccatttt
ggtgggtatgctgtttggac
gattatcattgccacgaccc
gggcaaaacagaggatttga
tcaccgttttgatgaagatcc
acggctttccatttgcatac
gcagcctaatgcgagattgt
cagtggtgataacctctcctg

tgacgaggtcatcagactgc
taatggcggctcttcagtct
ttctacatcaccacccctcc
ccaagtttctatgcttccgc
ggaaccgtttctgcatttgt
tccgatcgaaagaggaagaa
tccatcttgctcaactcacg
cgaccaactctgtaaaaccct
cgatgagctcacagcagaat
ggcattgcctttcattttgt(tc)33(ag)29(ag)25(ga)8g(ga)25(at)5(ta)25(ag)24(ct)24(ag)23(ag)23(ct)22

sloca
sloc
sloc
sloc
sloc
sloc
sloc
mloc
sloc
pres mloc

J6
J13
J5
J15

MEST016
MEST017
MEST019
MEST020
MEST021
MEST022
MEST023
MEST024
MEST026
MEST028
MEST029
MEST031
MEST032
MEST033
MEST034
MEST036
MEST037
MEST038

AB627195
AB627196
AB627197
AB627198
AB627199
AB627200
AB627201
AB627202
AB627203
AB627204
AB627205
AB627206
AB627207
AB627208
AB627209
AB627210
AB627211
AB627212

aaacaaaacaaaaggcacgg
aatagcctctgctgctctcg
caaacattcattgccaccag
caggccattagctccacattagttttggtggtttgcaagg
ggttggtattgggaccctct
aagggtatcattccattgcg
tgggatgtcaacctgcagta
caatctgctcatgttagctgc
attggcattgcttctcacct
atacaacccacgagtcccaa
tttcttctctcccaccctcc
ttgtttttgatgtacccttgc
tcaaacccccactagactcc
aaatcaatgatggaacggga
ttcggtttcggtaccatctc
tcattcgacacttcccaattc
tgcagtgacttgaatttggc

taactcatcccctcaccagc
ccgttaacccctcgtaatca
acttctcttgcctccgcata
gccactagcaaagttccctg
gcccaagactttgtcaccat
tcccaccaaaggagatgaag
cgatggactaaactgaatggc
aaaacatccctttcggcttt
gatgcacaacactcccacac
tgcaacaacaattcccttca
cagggaggctaaaatagggc
ggcatttcttgctcttctgc
ccaaaaatcgaaggaccaaa
caacaaagcagccataagca
cgaaggaccaaaaatggaga
gacctggcaaccttgtgaat
tagcatgcctggtctctcct
tcagccttctgttcctctctc(ag)21(ct)21(ga)21g(ga)6(ga)21(ga)21(ga)20(ga)20(ta)20(tc)20(ag)19(ag)19(ct)19(ct)19(ct)19(ct)19(ct)18(ct)18(ca)6(ga)18

sloc
sloc
mloc
sloc
sloc
sloc
sloc
mloc
mloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc

J11
J13
J8,J15
J17
J13
J15
J10
J2,J7
J15
J2
J17

MEST039
MEST041
MEST043
MEST044
MEST045
MEST049
MEST050
MEST052
MEST054
MEST056
MEST060
MEST061
MEST062
MEST063
MEST064
MEST065
MEST066
MEST067

AB627213
AB627214
AB627215
AB627216
AB627217
AB627218
AB627219
AB627220
AB627221
AB627222
AB627223
AB627224
AB627225
AB627226
AB627227
AB627228
AB627229
AB627230

ttgccactagagagccacac
tctcccatctcactttctcca
tgaggagttttatgggccag
ttcacccaaccgtagcaact
tggggacattggatggtatt
ccctcccacttctcgataca
gattatgaatgtcgtggccc
cggaccaacctacaaggcta
tgtgtgtgtgtgtgtgtgtga
agcttgaacctgtgctagatg
aagcaaccctccaaaaattc
tcgattcctcaatctctctca
caacacttgcccaagaacct
tcgctgaggtcaaaccttct
ttcagtttcaccatccctcc
aaaacccagacggagagaca
ttggcctctcctctcaaaaa
attttggtgggcagcaggt

tgcaacttttgccacatctc
cgaagatctgcctagccttg
gccaaccctttcttcctctt
ccctacgatttcgatgagga
gttgtcatggagccttggtt
ggatttcccgccatatttct
ccatgatcattcaagaccga
cacggatctacgactgagca
aagatgcgaaccgaagaaga
ttggctccctctaaaccgta
tggtaagcaagaaagccaca
atcggagaaaacccaaatcc
gccattttcagaacagccat
ggccattagctccacatcat
ggccattagctccacatcat
ttgtttgccttccaagctct
aaacatcttctgggcctgtg
aaaaacccaactctccgctt(ga)18(tc)18(ag)17(ag)17(at)17(tc)17(tg)17(ag)16(ag)16(at)16(ct)16(ct)16(ct)16(ct)16(ct)16(ct)16(ct)16ca(ct)8(tc)16sloc
pres mloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
pres mloc
sloc
sloc
sloc
sloc
sloc

J9
J14

J16
J12
J2
J15
J9

J9
J11
J9
J8
J7

J11
J4
J15
J13
J7
J6
J3
J8
J15
J9
J9
J5
J10
J2

‘Sanashi 63’

S13
S5
S15
S5
S12
S2,S7
S15

S13
S17
S15
S11
S8,S15
S2
S17
S3
S9
S9
S7
S10
S14
S10
S11

S15
S13
S7

S8
S15
S9
S5
S2

Tree Genetics & Genomes (2012) 8:709–723

713

Table 1 (continued)
Name

Accession

Forward primer
sequence

Reverse primer
sequence

Motif

Type ofmarker

MEST069
MEST070
MEST071
MEST073
MEST074
MEST076
MEST080
MEST082
MEST083
MEST084
MEST086
MEST088
MEST089
MEST091

AB627231
AB627232
AB627233
AB627234
AB627235
AB627236
AB627237
AB627238
AB627239
AB627240
AB627241
AB627242
AB627243
AB627244

cccgccaagttacaacctaa
ctctaacctacgctgctggg
cccaacaaacttcttctccg
ctctgaaactgcgaacacca
ttccgtcatcctcaaaggtc
aagtcccgaaagtgggttct
gctgcacttcttctgcattg
ctacctgagcctcctctccc
tttggggtttcaaagaggtg
caacggttttcattgagcag
cggaagtggaaagctgaaag
aaacagcaaaacccagatcg
tacagatttccgttcccctg
cggatcttttcttgaggtcg

cccaaaagaccaatgcttgt
tgtggacatcaagcttctgc
gctttccagctttgcgtaac
taaaccaaaaggctcacgct
gaggccatcagattttgcat
agacgcagcaaggttagagc
agttatggcctaatggcacg
ctcggaacggcaagaagtag
ctcaaatctccaaaccctgc
gctttcctcaagtcgtcgtc
gactgagtttcgcaagaggg
gggctctataaattccccca
aactccataacgcttcgctc
aacagatcgatcgtgaagcc(ct)15(ga)15(ga)15(tc)15(tc)15(ag)14(ct)14(ct)14(ga)14(ga)14(tc)14(ag)13(ag)13(ct)13

sloc
sloc
sloc
sloc
sloc
sloc
pres mloc
sloc
sloc
sloc
pres mloc
sloc
sloc
sloc

MEST092
MEST093
MEST095
MEST096
MEST097
MEST098
MEST099
MEST100
MEST101
MEST102
MEST105
MEST106
MEST108
MEST109
MEST111
MEST112
MEST113
MEST117

AB627245
AB627246
AB627247
AB627248
AB627249
AB627250
AB627251
AB627252
AB627253
AB627254
AB627255
AB627256
AB627257
AB627258
AB627259
AB627260
AB627261
AB627262

aaaatgtgtgacacccccat
tgctttctgcttaggcatctc
cctccaaatgctcatccatt
acaatcctcccccaaaactc
ttgatctgggaaaaatttgga
gcaacactgttcttgcggta
ccctccctccctctctctct
atgtctacggggtttgttgc
tgacatacccatctccccat
tcccggtctctttctgtctg
atattgaagggggcaccaa
catgatgcatggatctgctt
aaggaagtgtgtggcactcc
ggtgtaagttgtgtcgccaa
cacttccagatttccctcca
ctcactcaaaccctcccaaa
agagaaagggggacaaagga
cgaccttgaggcctctgtag

gagagagagggagtgaaggga
agacttctggtgttggggtg
tgcatcaagaggaagcagtg
cctcctcccccattcatatt
tttcacgctttgcaatgaag
ttgatagagagagaatgcggg
accaccccaatgcaaataat
tcatatcctttcttgcatttgtt
tgaggagggatttgaaggtg
attgttgaggcgaagtttgg
ggtgagaaacaaagtgggga
gcattgggagcattatcacc
ggctgtatgctgtatgatcagtg
actaacagcaccagcagcag
acaccaaacgaatgaggtcc
ctgctcatctcatcagccaa
agctcttggatctcggaaca
tccccatcaactactccgtc(ct)13(tc)5(ct)13(ct)13(ct)13(at)12(ag)25(at)11(cctc)5(ct)13(tg)12(tc)12(tc)12(ac)10(ta)12(ta)12(tc)11(ta)12(ta)12(at)12(tc)8tt(tc)11(ag)12(ag)12

mloc
sloc
mloc
sloc
sloc
pres mloc
pres mloc
mloc
sloc
sloc
sloc
pres mloc
pres mloc
sloc
sloc
mloc
pres mloc
mloc

MEST118
MEST119
MEST120
MEST122
MEST123
MEST124
MEST125
MEST126
MEST127
MEST128
MEST129
MEST132
MEST133
MEST134

AB627263
AB627264
AB627265
AB627266
AB627267
AB627268
AB627269
AB627270
AB627271
AB627272
AB627273
AB627274
AB627275
AB627276

gtggccaccacttgaatgta
tgcaacaggagaaaaccaga
agagagctgttttcgcttgg
catcacttgccctccaattt
caaacccttcgtcgcct
tccagggattacaatttcgg
ccatgagaaggcttggtgtt
tgaaaagatggcaggagctt
ggggtgaaggaggtttgaat
ttgcattcacttcaccatgc
gcacgagggagcaaagatttaaatcggctgatcactccc
agctctgaccctcctcaaca
ccttaaccgactgcctaacc

ggctactctcgacggctatg
tccaaatcaagaaactgccc
aatgctgaggcagtaatggg
tgcgacttaatgttgggtga
tgtctttgtctcggcaactg
tgcattggctcaagatgaag
cagagccgcattagggttt
ccaaagaaatcagaggcgaa
acgccagatgagaggctaaa
caagaagatggagatggtcaca
ttgatcttgcctgggttgat
ttggaaaacttccctcctca
agatttgggctttgaagtcg
tggggtgattctctggtttc(ag)12(at)11(ag)11(at)11(ct)11(ga)11(tc)11(at)11(ga)11(tc)11(ag)11(ta)11(tc)11(ct)11

sloc
sloc
sloc
sloc
sloc
pres mloc
sloc
sloc
sloc
mloc
pres mloc
sloc
sloc
sloc

Map position(linkage group)‘JM7’‘Sanashi 63’

J17
J11
J13
J9
J17
J3
J3
J15

S17

J12
J15
J13
J5
J4
J15
J13
J10
J17
J9
J16
J1
J11,J12
J8
J15

J10
J10
J12,J14J1
J3,J11
J11
J17
J1
J7
J5
J10
J15
J12
J15
J12
J5
J3

S9
S3
S3
S7
S12
S15
S13
S5
S11
S13
S5,S10
S17
S9

S8
S15
S2
S8
S15
S10
S12,S14
S11,S11
S17

S5
S10
S4
S15
S8
S1
S12
S5

714

Tree Genetics & Genomes (2012) 8:709–723

Table 1 (continued)
Name

Accession

Forward primer
sequence

Reverse primer
sequence

Motif

Type ofmarker

Map position(linkage group)‘JM7’

MEST135
MEST136
MEST138
MEST139
MEST140
MEST141
MEST142
MEST143
MEST144
MEST145
MEST147
MEST148
MEST150
MEST153

AB627277
AB627278
AB627279
AB627280
AB627281
AB627282
AB627283
AB627284
AB627285
AB627286
AB627287
AB627288
AB627289
AB627290

taggacagattgattccgcc
cctctgtacctaaccgcttcc
tttctcatggcagcttgatg
ggttgttgctgaccctcatt
cagcactgtgtgttacggct
tttctgagtgttgggttttgg
gttctccttggatcacctcg
ggaagtgggaggggagatag
catccaccatggaaagatcc
cgattcgggactcaaaattc
tccctccctcatcattcatc
gaggagagaaagtgggaggc
catcacttgcaagaagccaa
tttggaagcaaacgagaagc

ggggatttgtcacagcagtt
tcacctgggcagatttaagg
tctgcagtctggaagctcaa
tacgcttccgtaaaacccac
atgctgcgtttgtgtgtagg
gaaccccaaaacagagtgga
caattcagactccctctggc
tcttgctcaaccagcaacac
caggaaatgaaggagggtga
gataccagtgcggaggaaga
gcttgctcttggacttggac
tcctcccccaatgtcataaa
cttgtggtgtgtttcgatgg
gggaaactgatttagtgcgg(ta)11(ct)10(ct)10(ag)10(tc)10(ct)10(at)10(ag)10(ag)10(ag)10
(tc)9
(tc)9
(ct)9
(tc)9a(at)5

sloc
sloc
mloc
pres mloc
sloc
sloc
sloc
pres mloc
pres mloc
sloc
sloc
sloc
sloc
sloc

MEST154
MEST155
MEST157
MEST158
MEST159
MEST160
MEST161
MEST162
MEST163
MEST164
MEST165
MEST168
MEST170
MEST171
MEST174
MEST176
MEST177
MEST178

AB627291
AB627292
AB627293
AB627294
AB627295
AB627296
AB627297
AB627298
AB627299
AB627300
AB627301
AB627302
AB627303
AB627304
AB627305
AB627306
AB627307
AB627308

ataaaaacgacgtcatcggc
ttttcacagccaaacactgc
gtacggtgacaatggcgaat
gaaagacccgaaacccagat
tgcgtatagctcaaaacaagc
ttttccctctaaacaacctcca
cagagacatcagacagcgga
agcctctaacctaaccccca
gttttggagagaagcgaacg
cattttggtgatccggagag
tcacatctgctcttgctgct
taatggcggctcttcagtct
ttttaccccaaccctaaccc
aaacatgcagacagggaagg
cccaaaacccaactcagaaa
ctaaatccgtcactcccgaa
tttcgaatccgctgctactt
cccaggggatactctgtgaa

cccgggctagagagagaagt
gaacaaaggggaagaaaaaca
tccagaacgataggaatggc
tagggaaaattggtggcaaa
ggcatcgatgaagaagcag
ttgaaatggaacacgaaaacc
cagactccaaaacgacgaca
aaaaccgtcaattgctgtcc
gcttgaacttgctttagccac
tcgactgagatggtgagtgc
ggggaaaccacatgactctc
ccccgttaacccctcataat
gactcgggcactgtaattgg
ctgagcagtttgcagctttg
aagaatccccaatcccaatc
tgtgactggttttgtgggaa
agctcacgtgccttcttgat
tcgactgcacctgatgagac(tc)9(ct)8ttt(tc)5
(ct)9
(ct)9
(at)9
(ct)9ag(tc)5
(ag)9
(ct)9
(ga)9
(ac)9
(ag)9
(ct)9
(tctg)6(ggag)6ga(aggg)3(gaaa)5(ccag)5(agac)5(aaag)5

sloc
mloc
sloc
sloc
sloc
sloc
sloc
sloc
sloc
pres mloc
pres mloc
sloc
sloc
sloc
sloc
pres mloc
sloc
sloc

MEST182
MEST185
MEST187
MEST188
MEST189
MEST190
MEST193
MEST195
MEST196
MEST197
MEST198
MEST199
MEST200

AB627309
AB627310
AB627311
AB627312
AB627313
AB627314
AB627315
AB627316
AB627317
AB627318
AB627319
AB627320
AB627321

ccagcgagcagtacgga
agttggaatggctatgtgctc
acacacactccctcagaccc
gcacgatcagcagtttttca
atagggcaagagctcgacaa
tttgtgctcagacagatggc
attgttcacttggccaccat
ctgcctaaacaccaactcacc
aaaaacaagaagcagcagcc
gaacccggttttgatcctct
ccctttcagcaaagcaactc
gctcacaccactcttcctcc
acgccttcatccgtatcatc

gattggatggacggagaaga
ggcttcatttgcatttcctg
ccagctttctctggacttgg
tttcctttggtttcggagtg
ccacttgtgttctggtcacg
cattgatcatcaccactgcc
tccactgcaaccaacaagag
gagatcgtcaggggacaaaa
ttttcgaagtcctcccatct
tgacagacactcctccctcc
taccataactcgccggagtc
gttcaagagcctagcggatg
taaacaaaccaccgaaaccc(gaaa)4(tc)11(catt)4(agga)4(agaa)4(aatt)4(tat)10(tcc)10(ctc)10(gag)9(gca)9(cct)8(gtt)8pres mloc
mloc
sloc
sloc
sloc
sloc
sloc
pres mloc
slocpres mloc
mloc
sloc
sloc

a

sloc single locus, mloc multilocus, pres mloc presumed multilocus‘Sanashi 63’
S8

J16
J4,J12
J13
J11
J14

S4,S12

S2
J1
J16

S16
S9

J15
J15
J13
J12
J2,J15
J8
J13
J1
J6
J13
J1
J17
J15
J13
J16

S6
S15
S13
S12
S2
S1
S13
S1
S6
S13
S1

S13
S14

J13
J12
S9
J15
J10
J1,J7
J4
J8
J7

S1,S7
S4

S9
J1
J12
J8
J17
J2,J15
J1
J9

S13
S1
S9

Tree Genetics & Genomes (2012) 8:709–723

GeneScan software. AFLP marker names consist of thenames of primers, followed by the size of polymorphic
fragment in basepair.
Genotyping of the self-incompatibility locus
Genotypes at the self-incompatibility locus (S locus), which
is one of the important traits and has been mapped near thebottom of LG 17, were assessed by allele-specific PCR with
S3 and S9 allele-specific primer pairs (Broothaerts et al.
1995; Janssens et al. 1995). PCR amplification was performed as follows: initial denaturation at 94°C for 5 min,followed by for 35 cycles at 94°C for 1 min, 55°C for 1 min,and 72°C for 1 min. The PCR products were separated on a
1.5% agarose gel in 0.5× TBE buffer, stained with ethidium
bromide, and visualized under ultraviolet light.
Linkage analysis
We used a pseudo-testcross mapping strategy (Grattapaglia
and Sederoff 1994) to construct genetic linkage maps of‘JM7’ and ‘Sanashi 63’ with JoinMap v4.0 (van Ooijen
2006). This strategy allowed us to determine an accurate
marker order for the polymorphic loci within each of the twoparents separately. The regression mapping algorithm andthe Kosambi function in JoinMap v4.0 were applied. A
minimum LOD score of 8.0 was used to establish thelinkage groups. The two parental maps were aligned using
the markers segregating within both parents, and the linkage
groups were numbered using published apple reference
maps (Maliepaard et al. 1998; Liebhard et al. 2003) basedon SSR markers in common between the new maps andpreviously published maps.

715

Functional analysis of EST-SSRs
Characteristics of the EST-SSR sequences were identified bycomparisons to Arabidopsis thaliana protein sequences in
TAIR and the contigs from Apple v1.0 genome assembly. In
the BLASTX analysis, the amino acid sequences predictedfrom 106 of the EST-SSR sequences exhibited significant
homology to A. thaliana proteins (Supplementary Table 1).
Four EST-SSR sequences, MEST039, 140, 187, and 188,
exhibited high and significant homology to proteins related
to the Myb transcription factor. MEST124 showed high homology to a disease resistance protein of the TIR-NBS-LRR
class. In the BLASTN searches of contigs in Apple v1.0
genome assembly, all of the EST-SSR sequences tested in thisstudy showed very high homology to one or more apple
genome contigs, with E-values of under 2E-78 (Supplementary Table 2). The most homologous contigs (top hits) of 89
EST-SSRs were located in the same LGs as those identified by
mapping analysis (described in detail below), whereas theother 48 EST-SSRs were aligned to contigs in different LGs
than those predicted by mapping. When the search criteria
were expanded to include the top five most homologous
contigs for each EST (data not shown), at least one of the
top five contigs for 15 of these 48 ESTs corresponded to the
LG obtained in the mapping study.
AFLP analysis
AFLP analysis with 16 combinations of Mse and Eco primers yielded 189 segregating loci, including 125 for ‘JM7’
and 64 for ‘Sanashi 63’, with an average value of 7.8 and
4.0 loci per primer combination, respectively. The primer
combinations E32M59 and E33M48 generated the greatest
number of segregating loci (19), and E41M59 generated thefewest (4).

Results

Genetic linkage maps of ‘JM7’ and ‘Sanashi 63’

Development of EST-SSR markers

SSR markers from several published genetic linkage maps
were used to create genetic framework maps for both ‘JM7’
and ‘Sanashi 63’. Out of the 256 apple SSR markers tested,
155 revealed polymorphism in one or both parents and werepositioned into framework maps. The other 37 SSRs
showed polymorphism within one or both parents but were
not used for genotyping of F1 plants because they werelocated close to other mapped markers. The remaining 64
SSRs either did not show polymorphism or did not amplify
any products. For pear SSRs, 11 of 19 markers could bemapped in one or both parents. One of the pear markers
showed polymorphism within one parent but was not utilized for F1 genotyping because the marker located close toother mapped markers. The remaining seven pear markers

Out of the 9010 non-redundant EST sequences evaluated,
170, 9, and 21 EST sequences were selected for primer
design for di-, tri-, and tetranucleotide motifs, respectively.
PCR products with the desired target size were obtained
from 151, 9, and 21 primer pairs for di-, tri-, and tetranucleotide EST-SSRs, respectively. Finally, 137 EST-SSR
markers showed scorable heterozygous genotypes for atleast one parent, including 119 for di-, 7 for tri-, and 11
for tetranucleotide motifs (Table 1); 102 SSR primer pairs
generated amplified fragments from single loci, whereas 35
SSR primer pairs showed multilocus or presumed multilocus amplification.

Novel EST-SSR (numberof loci)Published SSR (numberof loci)
AFLP (number of loci)
Others (STS, cleaved
amplified polymorphic
sequence, and traits)(number of loci)
Total number of marker loci
Length of linkage groups(cM)
Marker density (markers/cM)

Marker type

Novel EST-SSR (numberof loci)Published SSR (numberof loci)
AFLP (number of loci)
Others (STS, cleaved
amplified polymorphic
sequence and traits)(number of loci)
Total number of marker loci
Length of linkage groups(cM)
Marker density (markers/cM)

Marker type

12
5
0

23
59.6
0.39

9

10
0

28
72.1

0.39

S2
6
16
5
2

29
59.7
0.49

S1

6

9

0
0

15
56.6

0.27

Linkage group

6

J2

9

J1

Linkage group

0.26

14
54.8

1
0

10

3

S3

0.52

28
53.5

8
0

15

5

J3

0.30

17
57.5

3
0

11

3

S4

0.26

22
84.0

6
0

12

4

J4

0.30

21
71.1

2
0

12

7

S5

0.43

29
67.8

9
0

15

5

J5

Table 2 Details of the linkage maps of ‘JM7’ (J) and ‘Sanashi 63’ (S)

0.20

12
59.3

3
0

7

2

S6

0.25

13
51.0

1
0

9

3

J6

0.23

14
62.1

4
0

5

5

S7

0.35

18
51.9

5
0

7

6

J7

0.45

19
41.8

3
0

9

7

S8

0.36

20
55.0

7
0

6

7

J8

0.25

15
58.9

2
0

4

9

S9

0.44

21
47.3

3
0

9

9

J9

0.21

13
61.8

2
0

6

5

S10

0.36

22
61.1

5
0

10

7

J10

0.38

24
63.3

9
0

10

5

S11

0.40

26
65.8

6
0

12

8

J11

0.49

12
24.7

2
0

4

6

S12

0.68

26
38.2

9
0

7

10

J12

0.36

21
57.8

2
0

9

10

S13

0.56

30
53.3

5
0

12

13

J13

0.30

14
47.1

2
0

9

3

S14

0.37

19
50.9

5
0

11

3

J14

0.32

29
90.5

6
0

12

11

S15

0.61

40
65.3

6
0

15

19

J15

0.36

19
53.5

9
0

9

1

S16

0.36

25
69.6

10
0

10

5

J16

0.36

22
61.2

4
1

12

5

S17

0.48

25
51.8

7
1

9

8

J17

0.32

310
981.8

59
3

154

94

Total

0.42

415
998.0

107
1

180

127

Total

716
Tree Genetics & Genomes (2012) 8:709–723

Tree Genetics & Genomes (2012) 8:709–723

either did not show polymorphism or did not amplify any
products.
In total, 137 newly developed EST-SSRs could be positioned on the ‘JM7’ or ‘Sanashi 63’ framework maps (Table 2; Fig. 1). Among them, 127 loci detected by 118
markers were mapped in ‘JM7’ and 94 loci detected by 86
markers were mapped in ‘Sanashi 63’.
AFLPs generated from 16 primer combinations were thenintegrated into these genetic linkage maps. An AFLP was
not placed if it made a change in the order of framework
SSRs, was more than 20 cM distal to the terminal SSR
marker of an LG, or showed severe distorted segregation
while neighboring markers did not. In total, 18 AFLP locifor ‘JM7’ and 6 for ‘Sanashi 63’ were excluded from themapping data files. As a result, AFLP analysis yielded 166
mapped loci, including 107 for ‘JM7’ and 59 for ‘Sanashi63’, with an average value of 6.3 and 3.5 loci per primer
combination, respectively (Table 2; Fig. 1).
The final genetic linkage maps of ‘JM7’ and ‘Sanashi 63’
consisted of 415 and 310 loci and spanned 998.0 and
981.8 cM, respectively (Table 2; Fig. 1). Total marker densities of the maps of ‘JM7’ and ‘Sanashi 63’ were 0.42 and0.32 markers per centimorgan, respectively (Table 2). These
two maps could be aligned by using 185 anchor SSR loci.
For the map of ‘JM7’, the length of the LGs ranged from
38.2 to 84.0 cM, with an average length of 58.7 cM. The
number of loci per LG in ‘JM7’ ranged from 13 to 40, withan average of 24.4. For the map of ‘Sanashi 63’, the lengthof the LGs ranged from 24.7 to 90.5 cM, with an average
length of 57.8 cM. The number of loci per LG ranged from12 to 29, with an average of 18.2. For both maps, marker
density was the highest in LG 12 and the lowest in LG 6.
Gaps between markers longer than 20 cM were observed in
one or both lines in LGs 1, 4, and 6. The largest gaps
between markers were located in LG 6:25.5 cM for ‘JM7’
and 22.4 cM for ‘Sanashi 63’, respectively. In both maps,
LG 15 contained the most SSR markers and LG 6 had thefewest. Compared with reference maps of ‘Discovery’ (M. ×domestica; Fig. 1) and ‘Fiesta’ (M. × domestica; notshown), new terminal SSR markers were obtained in thisstudy in the lower part of LG 7, the upper part of LG 8, andthe upper part of LG 13.
In this study, one published but previously unmapped
SSR marker could be mapped, and eight SSR markers were
mapped to LGs in addition to those reported in previously
published maps. Previously unmapped SSR marker NZ17e6(Guilford et al. 1997) was mapped in LG 17 of ‘Sanashi 63’.
SSR markers CH01e12 and CH01c06, both previously
reported to be located in LG 8, were also mapped here in
LG 1 of ‘Sanashi 63’. SSR marker Hi04b12, previously
reported to be located in LG 8, was also mapped here in
LG 15 of both ‘JM7’ and ‘Sanashi 63’. SSR markers
CH03b01 and NZmsEB107305, both reported to be in LG

717

2, were also mapped here in LG 15 of ‘JM7’ and LG 7 ofboth lines, respectively. Similarly, CH05c06, previously
reported to be in LG 16, was also mapped here in LG 13
of both ‘JM7’ and ‘Sanashi 63’, and CH05g11 andMDAJ761 (AJ000761-SSR), reported to be in LG 14, werealso mapped in LG 6 of ‘JM7’ and/or ‘Sanashi 63’.
Three markers previously mapped to only a single locus
mapped to several new loci in our mapping population.
Hi05g12, previously reported in LG 2, was also mapped
here in three new LGs (LG 10, 12, 14), all in ‘JM7’.
Hi07b02, previously reported in LG 4, was mapped here
in three new LGs (LG 7, 13, 17), and NZmsEB177464,
previously reported in LG 3, was mapped here in four LGs(LG 3, 10, 11, 17), sometimes in several locations per LG.
Three SSR markers, GD103, CH05b06, and Hi04c10,
which were previously mapped at two loci, were mapped
here at two or more new loci. GD103 revealed two new
segregating loci in LGs 4 and 14 in addition to its previously
reported locations in LGs 5 and 10; CH05b06 revealed two
new segregating loci in LGs 15 and 17 in addition to its
previously reported locations in LGs 5, 10, and 16.
Hi04c10, previously mapped in LGs 3 and 4, generated five
new segregating loci in LGs 2, 3 (a second locus was
mapped in ‘JM7’), 4 (a second locus was mapped in ‘Sanashi 63’), 5, and 13.

Discussion
The genetic linkage map of ‘Sanashi 63’ is the first genetic
linkage map covering all 17 LGs in a species belonging to
the section Sorbomalus, and it was constructed primarily
with informative codominant SSR markers. Although
genetic linkage maps of the genus Malus have been constructed with SSR markers covering all 17 LGs (Maliepaard
et al. 1998; Silfverberg-Dilworth et al. 2006; FernandezFernandez et al. 2008; van Dyk et al. 2010), these maps
were intended primarily for the fruiting scion apple (M. ×domestica). The only genetic linkage maps of Malus spp.
other than M. domestica have been those of rootstocks ‘M.9’
and ‘R.5’ (Celton et al. 2009). Because ‘JM7’ is a descendant of ‘M.9’ and M. prunifolia, ‘JM7’ would be expected
to possess a similar genetic background to ‘M.9’. However,M. sieboldii, to which ‘Sanashi 63’ belongs, is categorized
in section Sorbomalus rather than in section Malus where
M. × domestica is classified. The section Sorbomalus includes
Malus florentina, Malus kansuensis, and Malus yunnanensis, and none of which have yet been used in any mapping
project.
Overall, a total of 300 SSRs were mapped onto the ‘JM7’
and ‘Sanashi 63’ genetic maps, which were then comparedwith those of ‘Discovery’ and ‘Fiesta’ (both M. × domestica). In most cases, the length of each LG was shorter than

718

Fig. 1 Genetic maps of ‘JM7’, ‘Sanashi 63’, and ‘Discovery’ (SilfverbergDilworth et al. 2006). Linkage groups are designated as J1 to J17 for ‘JM7’,
S1 to S17 for ‘Sanashi 63’ and D1 to D17 for ‘Discovery’. Only terminal SSR
markers and anchor SSR markers are indicated in the map of ‘Discovery’.

Tree Genetics & Genomes (2012) 8:709–723

Numbering of the linkage group is according to Maliepaard et al. (1998).
Genetic distances and marker names are listed next to each linkage group bar.
Names of novel apple EST-SSRs are underlined. Names of published apple
SSRs and pear SSRs are indicated in bold and italic font, respectively

Tree Genetics & Genomes (2012) 8:709–723

Fig. 1 (continued)

719

720

Fig. 1 (continued)

Tree Genetics & Genomes (2012) 8:709–723

Tree Genetics & Genomes (2012) 8:709–723

those of the reference maps; however, the differences are noteasily explained. Some incomplete regions were observed at
the bottom of LG 12, the top of ‘Sanashi 63’ LG 1, thebottom of ‘Sanashi 63’ LG 3, the bottom of ‘Sanashi 63’ LG
9, and the top of ‘JM7’ LG 11. In these regions, the tested
markers could be amplified but did not show polymorphism.
Despite the lack of map information in these regions, thegenome coverage of the maps of ‘JM7’ and ‘Sanashi 63’ iscomparable to that of the reference map of ‘Discovery’(Silfverberg-Dilworth et al. 2006) because three otherregions (LGs 7, 8, and 13) were expanded in this study.
The marker density in the maps of ‘JM7’ (0.42 markers/cM)
and ‘Sanashi 63’ (0.32 markers/cM; Table 2) were markedly
higher than those of ‘M.9’ (0.16 markers/cM) and ‘R.5’(0.19 markers/cM). Additionally, the ‘JM7’ and ‘Sanashi
63’ maps also showed higher genome coverage than ‘M.9’
and ‘R.5’.
Considering the genetic maps of ‘Discovery’, ‘JM7’, and‘Sanashi 63’ as representative of domesticated and wild
apples in sections Malus and Sorbomalus, it is possible tocompare the genome structures among those apples. The
marker order in each of the three maps was almost the same,
indicating that macrosynteny is retained among Malus spp.
Six of the 14 SSRs that mapped in different LGs frompreviously published maps were located in suggested orthologous LGs (chromosomes) and are presumed to have originated from a genome-wide duplication event (Velasco et al.
2010). Differences between the materials used here andthose in previous studies enabled us to map previously
unmapped loci. However, eight SSRs were mapped on
chromosomes that were inconsistent with the current modelof genome-wide duplication in apple. Because most of these
SSRs showed three or more amplified bands, these markers
may have detected heavily duplicated regions unrelated togenome-wide duplication. SSR marker CH01c06 amplified
bands in ‘JM7’ on LG 8; however, additional bands thatmapped to LG 1 was amplified in ‘Sanashi 63’, possibly
suggesting a short block of chromosome rearrangement.
A relatively small number of EST-SSRs have been available until now, and their development is still an important
objective. Fewer than 200 EST-SSRs derived from apple
were developed and mapped in previous reports (Celton etal. 2009; Silfverberg-Dilworth et al. 2006; van Dyk et al.
2010), whereas approximately 300 genomic SSRs havebeen reported. An advantage of EST-SSRs is that they areoften transferable to other rosaceous crops (Gasic et al.
2009b); thus, development of EST-SSRs in one species
can directly benefit genomic studies in another. The ESTSSR markers described here were designed from M. ×domestica sequences because we assumed that thesemarkers would be applicable not only to rootstocks but also
to M. × domestica cultivars. The findings that many of themarkers were successfully mapped in ‘JM7’ or ‘Sanashi 63’

721

suggests that these EST-SSRs will be useful within the
broad Malus collections for accurate and high-throughput
genotyping and may be candidates for selection markers for
MAS.
The newly developed EST-SSR markers in our study
showed higher polymorphism than those reported in previous studies. The proportion of successfully mapped SSR
markers was 71% for dinucleotide motifs, whereas 66%
was the highest proportion in other reports (van Dyk et al.
2010). Tri- and tetranucleotide motifs also showed higher
polymorphism than in previous reports (Celton et al. 2009;van Dyk et al. 2010). These results revealed higher heterozygosity in the genomes of ‘JM7’ and ‘Sanashi 63’ than in
the cultivated apples for which genetic maps had previously
been published. These differences may be explained by ouruse of interspecific hybrids between wild accessions forgenome mapping. Thus, the genetic maps of ‘JM7’ and‘Sanashi 63’ and the mapping populations reported here
may be useful to identify the locations of new markers forwhich the map locations are unknown.
Fourteen of the novel EST-SSRs reported here mapped tomultiple loci. The combinations of multiply mapped loci(chromosomes) amplified by one marker were generally
consistent with combinations of orthologous chromosomes
predicted by a model of genome-wide duplication (Velasco
et al. 2010). Markers MEST024 and MEST198 amplified
bands corresponding to two loci in ‘JM7’ and one locus in‘Sanashi 63’. The two loci detected by each of these twomarkers in ‘JM7’ were mapped in LGs consistent withgenome-wide duplication. However, the map locations for
MEST024 and MEST198 in ‘Sanashi 63’ were different
from those of ‘JM7’ and not readily explained by genome
wide duplication, which might suggest a short block ofchromosome rearrangement.
When BLASTN searches were performed using the single most homologous contigs in Apple v1.0 genome assembly, 48 EST-SSRs were aligned to contigs in different LGs
than those predicted by mapping. When the criteria for
BLASTN searches of EST-SSRs were then expanded toinclude the second- to fifth-most homologous contigs withinApple v1.0 genome assembly, at least one of the top fivecontigs for 15 of these 48 EST-SSRs corresponded to the
LG obtained by mapping, suggesting that these ESTs are
members of gene families that are widely scattered in the
genome. However, the remaining 33 markers still showeddiscrepancies between the LG of the most homologous
contigs and the LG obtained by mapping. Possible reasonsfor this could be misassembly of ESTs, gene families consisting of genes possessing similar sequences, and misassembly of contigs in Apple v1.0 genome sequence. These
results indicate that BLAST homology searching against
Apple v1.0 genome assembly is not always the best approach to estimate the correct map location(s) of a nucleic

722

acid sequence. Thus, mapping approaches using genetic
linkage maps with broad genome coverage are necessary
for determining the correct map location(s) of a sequence.
Additionally, novel EST-SSRs that were aligned within unanchored metacontigs by BLAST searching were useful inassigning the map positions of those metacontigs within
LGs (chromosomes).
The newly constructed maps of ‘JM7’ and ‘Sanashi 63’
are expected to provide effective tools for apple rootstock
breeding. Apple rootstock breeding has traditionally been
conducted by selection of seedlings from wild populationsof apples (Webster and Wertheim 2003). Although modern
breeders introduced controlled crossing techniques to replace open-pollinated seedling selection; to date, all newrootstocks have been selected using conventional phenotypic evaluation techniques, which are time-consuming andlabor-intensive activities. These obstacles can be overcome
by using molecular markers linked to important traits. MAS
will enable apple breeders to achieve dramatic progress inrootstock breeding programs more quickly and accurately
than before. The linkage maps of ‘JM7’ and ‘Sanashi 63’
will be worthwhile for analyzing distinctive and importantcharacteristics, such as easy propagation by cutting andresistance to diseases such as crown gall (Moriya et al.
2010) and Valsa canker (Abe et al. 2011). Thus, the genetic
maps of ‘JM7’ and ‘Sanashi 63’ provide a development
platform for assessing inheritance patterns and direct adoption of selection markers for these traits. The major goal of
our study was the development of excellent new rootstocks
based on introgression of biotic stress resistance into superior rootstock cultivars. The next step to facilitate rootstock
breeding by an MAS system is to identify and map lociinvolved in traits of interest.
Acknowledgments We thank Dr. T. Ueda of NARO Institute of Fruit
Tree Science for valuable discussions and computer analysis. This
work was supported by National Agriculture and Food Research Organization Research Project No. 211, “Establishment of Integrated
Basis for Development and Application of Advanced Tools for DNA
Marker-Assisted Selection in Horticultural Crops”.

References
Abe K, Kotoda N, Kato H, Soejima J (2011) Genetic studies on
resistance to Valsa canker in apple: genetic variance and breedingvalues estimated from intra- and inter-specific hybrid progeny
populations. Tree Genet Genomes 7:363–372
Bonfield JK, Smith KF, Staden R (1995) A new DNA sequenceassembly program. Nucleic Acids Res 23:4992–4999
Broothaerts W, Janssens GA, Proost P, Broekaert WF (1995) cDNA
cloning and molecular analysis of two self-incompatibility alleles
from apple. Plant Mol Biol 27:499–511
Bus VGM, Chagne D, Bassett HCM, Bowatte D, Calenge F, Celton
JM, Durel CE, Malone MT, Patocchi A, Ranatunga AC, Rikkerink EHA, Tustin DS, Zhou J, Gardiner SE (2008) Genome

Tree Genetics & Genomes (2012) 8:709–723
mapping of three major resistance genes to woolly apple aphid(Eriosoma lanigerum Hausm.). Tree Genet Genomes 4:223–236
Celton JM, Tustin DS, Chagne D, Gardiner SE (2009) Construction ofa dense genetic linkage map for apple rootstocks using SSRs
developed from Malus ESTs and Pyrus genomic sequences. Tree
Genet Genomes 5:93–107
Costa F, van de Weg WE, Stella S, Dondini L, Pratesi D, Musacchi S,
Sansavini S (2008) Map position and functional allelic diversityof Md-Exp7, a new putative expansin gene associated with fruit
softening in apple (Malus × domestica Borkh.) and pear (Pyrus
communis). Tree Genet Genomes 4:575–586
Decroocq V, Fave MG, Hagen L, Bordenave L, Decroocq S (2003)
Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor Appl Genet 106:912–922
Fernandez-Fernandez F, Evans KM, Clarke JB, Govan CL, James CM,
Maric S, Tobutt KR (2008) Development of an STS map of aninterspecific progeny of Malus. Tree Genet Genomes 4:469–479
Gasic K, Gonzalez DO, Thimmapuram J, Liu L, Malnoy M, Gong G,
Han Y, Vodkin LO, Aldwinckle HS, Carroll NJ, Orvis KS, Goldsbrough P, Clifton S, Pape D, Fulton L, Martin J, Theising B,
Wisniewski ME, Fazio G, Feltus FA, Korban SS (2009a) Comparative analysis and functional annotation of a large expressedsequence tag collection of apple. Plant Genome 2:23–38
Gasic K, Han YP, Kertbundit S, Shulaev V, Iezzoni AF, Stover EW,
Bell RL, Wisniewski ME, Korban SS (2009b) Characteristics andtransferability of new apple EST-derived SSRs to other Rosaceae
species. Mol Breed 23:397–411
Gianfranceschi L, Seglias N, Tarchini R, Komjanc M, Gessler C (1998)
Simple sequence repeats for the genetic analysis of apple. Theor
Appl Genet 96:1069–1076
Grattapaglia D, Sederoff R (1994) Genetic-linkage maps of Eucalyptus
grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics 137:1121–1137
Guilford P, Prakash S, Zhu JM, Rikkerink E, Gardiner S, Bassett H,
Forster R (1997) Microsatellites in Malus × domestica (apple):abundance, polymorphism and cultivar identification. Theor Appl
Genet 94:249–254
Hatton RG (1917) Paradise apple rootstocks. J Royal Hort Soc 42:361–
399
Hemmat M, Weeden NF, Brown SK (2003) Mapping and evaluation ofMalus × domestica microsatellites in apple and pear. J Amer Soc
Hort Sci 128:515–520
Hokanson SC, Szewc-McFadden K, Lamboy WF, McFerson JR (1998)
Microsatellite (SSR) markers reveal genetic identities, genetic
diversity and relationships in a Malus × domestica Borkh. core
subset collection. Theor Appl Genet 97:671–683
Janssens GA, Goderis IJ, Broekaert WF, Broothaerts W (1995) A
molecular method for S-allele identification in apple based onallele-specific PCR. Theor Appl Genet 91:691–698
Kotoda N, Wada M, Komori S, Kidou S, Abe K, Masuda T, Soejima J(2000) Expression pattern of homologues of floral meristem identity genes LFY and AP1 during flower development in apple. J
Amer Soc Hort Sci 125:398–403
Liebhard R, Gianfranceschi L, Koller B, Ryder CD, Tarchini R, Van deWeg E, Gessler C (2002) Development and characterisation of
140 new microsatellites in apple (Malus × domestica Borkh.).
Mol Breed 10:217–241
Liebhard R, Koller B, Gianfranceschi L, Gessler C (2003) Creating asaturated reference map for the apple (Malus × domestica Borkh.)
genome. Theor Appl Genet 106:1497–1508
Luby JJ (2003) Taxonomic classification and brief history. In: Ferree
DC, Warrington IJ (eds) Apples botany, production and uses.
CABI Publishing, Cambridge, pp 1–14
Maliepaard C, Alston FH, Van Arkel G, Brown LM, Chevreau E,
Dunemann F, Evans KM, Gardiner S, Guilford P, Van Heusden
AW, Janse J, Laurens F, Lynn JR, Manganaris AG, Nijs APMd,

Tree Genetics & Genomes (2012) 8:709–723
Periam N, Rikkerink E, Roche P, Ryder C, Sansavini S, Schmidt
H, Tartarini S, Verhaegh JJ, Van Ginkel MV, King GJ (1998)
Aligning male and female linkage maps of apple (Malus pumila
Mill.) using multi-allelic markers. Theor Appl Genet 97:60–73
Moriya S, Iwanami H, Takahashi S, Kotoda N, Suzaki K, Abe K(2008) Evaluation and inheritance of crown gall resistance inapple rootstocks. J Jpn Soc Hort Sci 77:236–241
Moriya S, Iwanami H, Takahashi S, Kotoda N, Suzaki K, Yamamoto T,
Abe K (2010) Genetic mapping of the crown gall resistance gene of
the wild apple Malus sieboldii. Tree Genet Genomes 6:195–203
Moriya S, Iwanami H, Okada K, Yamamoto T, Abe K (2011) A practicalmethod for apple cultivar identification and parent–offspring analysis using simple sequence repeat markers. Euphytica 177:135–150
Naik S, Hampson C, Gasic K, Bakkeren G, Korban SS (2006) Development and linkage mapping of E-STS and RGA markers forfunctional gene homologues in apple. Genome 49:959–968
Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EHA, Allan AC,
Beuning LL, Bowen JH, Gera E, Jamieson KR, Janssen BJ, Laing
WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJF,
Walton EF, Yauk YK (2006) Analyses of expressed sequence tags
from apple. Plant Physiol 141:147–166
Nishitani C, Terakami S, Sawamura Y, Takada N, Yamamoto T (2009)
Development of novel EST-SSR markers derived from Japanesepear (Pyrus pyrifolia). Breed Sci 59:391–400
Phipps JB, Robertson KR, Smith PG, Rohrer JR (1990) A checklist of
the subfamily Maloideae (Rosaceae). Can J Bot 68:2209–2269
Pilcher RLR, Celton JM, Gardiner SE, Tustin DS (2008) Genetic
markers linked to the dwarfing trait of apple rootstock ‘Malling
9’. J Am Soc Hort Sci 133:100–106
Robinson T, Fazio G, Holleran T, Aldwinckle H (2003) The Geneva
series of apple rootstocks from Cornell: performance, disease
resistance, and commercialization. Acta Hort 622:513–519
Sawamura Y, Saito T, Takada N, Yamamoto T, Kimura T, Hayashi T,
Kotobuki K (2004) Identification of parentage of Japanese pear
‘Housui’. J Jpn Soc Hort Sci 73:511–518
Silfverberg-Dilworth E, Matasci CL, Van de Weg WE, Van Kaauwen
MPW, Walser M, Kodde LP, Soglio V, Gianfranceschi L, Durel
CE, Costa F, Yamamoto T, Koller B, Gessler C, Patocchi A (2006)
Microsatellite markers spanning the apple (Malus × domestica
Borkh.) genome. Tree Genet Genomes 2:202–224

723
Soejima J, Yoshida Y, Haniuda T, Bessho H, Tsuchiya S, Masuda T,
Komori S, Sanada T, Ito Y, Sadamori S, Kashimura Y (2010) New
dwarfing apple rootstocks ‘JM1’, ‘JM7’ and ‘JM8’. Bull Natl Inst
Fruit Tree Sci 11:1–16
van Dyk MM, Soeker MK, Labuschagne IF, Rees DJG (2010) Identification of a major QTL for time of initial vegetative budbreak inapple (Malus × domestica Borkh.). Tree Genet Genomes 6:489–
502
van Ooijen JW (2006) JoinMap 4 Software for the calculation ofgenetic linkage maps in experimental populations. Kyazma B.V.,
Wageningen, Netherlands
Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman
A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M,
Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V,
Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P,
Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M,
Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin
N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira
C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D,
Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q,
Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A,
Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan
AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E,
Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B,
Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE,
Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R(2010) The genome of the domesticated apple (Malus × domestica
Borkh.). Nat Genet. doi:10.1038/ng.654
Webster AD, Wertheim SJ (2003) Apple rootstocks. In: Ferree DC,
Warrington IJ (eds) Apples botany, production and uses. CABI
Publishing, Cambridge, pp 91–124
Yamamoto T, Kimura T, Shoda M, Ban Y, Hayashi T, Matsuta N(2002a) Development of microsatellite markers in Japanese pear.
Mol Ecol Notes 2:14–16
Yamamoto T, Kimura T, Shoda M, Imai T, Saito T, Sawamura Y,
Kotobuki K, Hayashi T, Matsuta N (2002b) Genetic linkage maps
constructed by using an interspecific cross between Japanese andEuropean pears. Theor Appl Genet 106:9–18
Zagaja SW (1980) Performance of two apple cultivars on P. series
dwarf rootstocks. Acta Hort 114:162–169