Tree Genetics & Genomes (2006) 2: 202–224DOI 10.1007/s11295-006-0045-1

ORIGINAL PAPER

E. Silfverberg-Dilworth . C. L. Matasci .W. E. Van de Weg . M. P. W. Van Kaauwen .M. Walser . L. P. Kodde . V. Soglio . L. Gianfranceschi .C. E. Durel . F. Costa . T. Yamamoto . B. Koller .C. Gessler . A. Patocchi

Microsatellite markers spanning the apple (Malus x domesticaBorkh.) genomeReceived: 19 December 2005 / Revised: 27 April 2006 / Accepted: 18 May 2006 / Published online: 9 August 2006# Springer-Verlag 2006

Abstract A new set of 148 apple microsatellite markershas been developed and mapped on the apple referencelinkage map Fiesta x Discovery. One-hundred andseventeen markers were developed from genomic

libraries enriched with the repeats GA, GT, AAG,AAC and ATC; 31 were developed from EST sequences.Markers derived from sequences containing dinucleotiderepeats were generally more polymorphic than sequencescontaining trinucleotide repeats. Additional eight SSRsfrom published apple, pear, and Sorbus torminalis SSRs,whose position on the apple genome was unknown, havealso been mapped. The transferability of SSRs acrossMaloideae species resulted in being efficient with 41%of the markers successfully transferred. For all 156SSRs, the primer sequences, repeat type, map position,and quality of the amplification products are reported.Also presented are allele sizes, ranges, and number ofSSRs found in a set of nine cultivars. All thisinformation and those of the previous CH-SSR seriescan be searched at the apple SSR database (http://www.hidras.unimi.it) to which updates and comments can beadded. A large number of apple ESTs containing SSRrepeats are available and should be used for thedevelopment of new apple SSRs. The apple SSRdatabase is also meant to become an internationalplatform for coordinating this effort. The increasedcoverage of the apple genome with SSRs allowed theselection of a set of 86 reliable, highly polymorphic, andoverall the apple genome well-scattered SSRs. TheseSSRs cover about 85% of the genome with an averagedistance of one marker per 15 cM.

Keywords SSR . Genetic mapping .Simple sequence repeat

Introduction

In the last few years, apple genetics has made significantprogresses, partly due to the increasing availability ofmulti-allelic SSR markers. These markers proved to beextremely useful for integrating mapping results fromindependent studies and in the development of innovativeprocedures for assessing marker–gene associations. Theirhigh level of transferability, general high level of poly-

E. Silfverberg-Dilworth and C. L. Matasci contributed equally tothis work.

E. Silfverberg-Dilworth . C. L. Matasci . M. Walser .C. Gessler . A. PatocchiPlant Pathology,Institute of Integrative Biology (IBZ), ETH Zurich,CH-8092 Zurich, Switzerland

W. E. Van de Weg . M. P. W. Van Kaauwen . L. P. KoddeDepartment of Biodiversity and Breeding,Plant Research International,P.O. Box 16, 6700 AA Wageningen, The Netherlands

V. Soglio . L. GianfranceschiDepartment of Biomolecular Sciences and Biotechnology,University of Milan,Via Celoria 26,20133 Milan, Italy

C. E. DurelGenetics and Horticulture (GenHort),National Institute for Agronomical Research (INRA),BP 60057, 49071 Beaucouzé Cedex, France

F. CostaDepartment of Fruit Tree and Woody Plant Sciences,University of Bologna,40127 Bologna, Italy

T. YamamotoNational Institute of Fruit Tree Science,Tsukuba Ibaraki 305-8605, Japan

B. KollerEcogenics GmbH,Wagistrasse 23,CH-8952 Zurich-Schlieren, Switzerland

A. Patocchi (*)LFW C16,Universitätstrasse 2,CH-8092 Zürich, Switzerlande-mail: andrea.patocchi@agrl.ethz.ch

morphism, and the relative ease by which they aregenerated, being PCR-based, makes them the marker ofchoice for alignments among linkage maps of apple. As aresult, recent maps have been based on a backbone ofmulti-allelic SSR markers embedded in RAPD and/orAFLP markers, which can be produced in large amounts ina relatively short time (Liebhard et al. 2002, 2003b; Kenisand Keulemans 2005). Alignments can be made for theidentification and orientation of corresponding homolo-gous linkage groups as well as for relative positions ofspecific loci.

SSR markers are, therefore, extremely valuable forbuilding integrated genetic maps comprising genes,confidence intervals of QTLs, and other loci gatheredfrom multiple maps. SSR-based maps have been essentialfor identification and positional comparison of major genesand QTLs for scab, powdery mildew, and fire blightresistance (Durel et al. 2000, 2003; Evans and James 2003;Liebhard et al. 2003c; Calenge et al. 2004; Gygax et al.2004; James et al. 2004; Patocchi et al. 2004; Tartarini et al.2004; Vinatzer et al. 2004; Calenge et al. 2005; Calengeand Durel 2005; Khan et al. 2006) as well as morphologicalor physiological traits (Conner et al. 1998; King et al. 2000;Liebhard et al. 2003a; Costa et al. 2005). Recognition ofSSR associations led to the identification of clusters ofresistance genes (e.g., Bus et al. 2004) and to the firstintegrated map of apple that reviews many papers withregard to the location of scab resistance genes (Durel et al.2004). The recent discovery of many new gene–SSRmarker associations, made necessary a new update of thisintegrated map with recently mapped scab resistance genes(Bus et al. 2005a,b; Patocchi et al. 2005), thus continuouslyimproving our understanding of the organization of theapple genome.

Technically, SSR markers are highly suitable for directgenotyping of any new individual, being easily transfer-able, multi-allelic, and having a known map position. Theyare also the most cost effective marker for directedgenome-wide genotyping approaches, as with knownmap positions only a relatively low number of well-selected markers have to be tested to obtain a goodcoverage. The possibility of multiplexing several SSRmarkers in the same PCR reaction allows an additionalreduction of the costs of genotyping.

A good coverage of the apple genome with SSR markersis the prerequisite for two innovative techniques forassessment of molecular-marker trait associations. Firstly,the Genome Scanning Approach (GSA, Patocchi andGessler 2003) allows efficient mapping of major genes.This procedure was successfully applied to map the applescab resistance genes Vr2, Vm (Patocchi et al. 2004;Patocchi et al. 2005), and Vb (Erdin et al. 2006). Secondly,the Pedigree Genotyping concept was developed (Van deWeg et al. 2004), which allows the exploitation of breedingmaterial in the assessment of marker–trait associations andin allele mining by using multiple pedigreed plantpopulations, which can be any combination of crosses,cultivars, and breeding lines. This concept makes use ofdirected genotyping and the so-called Identity By Descent

(IBD) concept. It forms the base of the EU-HiDRASproject (Gianfranceschi and Soglio 2004) aimed at a proofof concept for Pedigree Genotyping and at the identifica-tion of molecular markers for fruit quality and diseaseresistance.

The major disadvantage in the use of SSR markers is theconsiderable initial investment needed to develop and mapthem. Although around 160 SSRs have been developed forthe apple (Guilford et al. 1997; Gianfranceschi et al. 1998;Hokanson et al. 1998; Liebhard et al. 2002; Hemmat et al.2003; Vinatzer et al. 2004), their distribution within thegenome is not homogenous. Almost all linkage groupscontain regions with large gaps between two successiveSSRs (Liebhard et al. 2003b). The development of newSSRs may solve this problem. Thus far, the most widelyused method to produce SSRs is based on the cloning andsequencing of genomic fragments enriched for a repeatedsequence and the designing of upstream and downstreamprimers (Tenzer et al. 1999; Gautschi et al. 2000).Additionally, some new SSR markers have been obtainedby selecting the transferable SSR markers from closelyrelated species (Yamamoto et al. 2004).

Recently, apple genomic projects have made thou-sands of apple EST sequences available, which can nowbe searched for SSR repeats and used for the develop-ment of new SSR markers (Crowhurst et al. 2005;Korban et al. 2005). This approach has severaladvantages: 1) no enriched genomic library has to beconstructed; 2) extensive sequencing is not necessary, thusreducing the cost of the development of the SSRmarkers; 3)it is possible to develop SSRmarkers for which it is difficultto construct enriched libraries (e.g., AT repeats), and last butnot least, 4) markers are developed from coding sequences.

In this paper, we present a new, extensive set of appleSSRs, developed within the framework of the HiDRASEuropean project (Gianfranceschi and Soglio 2004), fromgenomic libraries, publicly available EST sequences, andSSR markers of other species closely related to Malus. Allthese SSRs are also tested for their level of polymorphismand are positioned on a molecular marker linkage map.These SSRs, together with those already published, havebeen used to select a set of 86 highly polymorph SSRswell-scattered on the apple genome.

Materials and methods

Plant material and DNA extraction

Cultivars Elstar, Golden Delicious, and Florina were usedto construct the SSR libraries. A series of nine diploidcultivars (Fiesta, Discovery, Florina, Nova Easygro, TN10-8, Durello di Forlì, Prima, Mondial Gala, and Fuji) wasused to estimate the level of polymorphism of the newmarkers. Forty-four progeny plants of the Fiesta ×Discovery cross, which is a subset of the 251 plantsused by Liebhard et al. (2003b) to generate “the referencemap”, were used to map the new SSRs. Three othermapping populations, Discovery × TN10-8 (149 plants),

203

Durello di Forlì × Fiesta (subset of 60 plants), or Fuji ×Mondial Gala (subset of 60 plants), were used to map theSSRs that could not be mapped in the cross Fiesta ×Discovery. DNA was extracted according to Koller et al.(2000), gel quantified and diluted to 1 ng/μl.

SSR development

Genomic libraries

SSR-enriched libraries from Elstar were developed at PlantResearch International. The procedure for microsatelliteenrichment by selective hybridization was modified fromKaragyozov et al. (1993) by Van de Wiel et al. (1999) andVan der Schoot et al. (2000). DNAwas digested with TaqIand size-fractionated by agarose gel electrophoresis. Frag-ments between 300 and 1,000 bp were recovered byelectro-elution, enriched by hybridization to five oligonu-cleotides (GA, GT, ATC, AAG, ACC), ligated in pGEM-T(Promega) or pCRII-TOPO (Invitrogen) and transformedto competent TOP10 F’ (Invitrogen). Colonies weretransferred onto Hybond N+ membranes and hybridizedwith the appropriately labeled oligonucleotides. Positiveclones were sequenced with the primers Sp6 and T7 byGreenomics™ (Wageningen, the Netherlands). The en-riched libraries of Golden Delicious (ATC) and Florina(AAG and AAC) were developed by Ecogenics GmbH(Zürich, Switzerland) from size-selected digested (MboIfor AAG and AAC libraries and Tsp509I for the ATClibrary) genomic DNA ligated to adaptors and enriched bymagnetic bead selection with biotin-labeled correspondingoligonucleotide repeats (Gautschi et al. 2000). DNAfragments were PCR-amplified with the correspondingprimer (Table 1). The PCR products of the ATC librarywere cloned into the vector Topo® (Invitrogen) andtransformed in the TOP10 F’ competent cells (Invitrogen),while the AAC and AAG libraries were cloned in thevector pDrive (Qiagen) and transformed in the EZ cells(Qiagen). Recombinant cells were spotted over nylonmembranes and hybridized with the corresponding SSRrepeat. Positive clones were sequenced with the primerM13 reverse by Synergene Biotech GmbH (Zürich,Switzerland).

SSRs from publicly available ESTs

Malus sequences from the NCBI database (September2003) (http://www.ncbi.nlm.nih.gov/) were examined formicrosatellite repeats using the software Tandem RepeatFinder v 3.21 (Benson 1999). From these ESTs, a subset ofsequences was selected that contained microsatelliterepeats and in which the repeat was sufficiently far fromthe edge of the sequence to allow design of both forwardand reverse PCR primers.

SSRs from the literature

Eight apple SSRs with unknownmap position (GD12, -15,-96, -100, -103, -142, -147, and -162; Hokanson et al.1998) have been tested for polymorphism with andbetween Fiesta and Discovery as well as with the parentsof the other mapping populations available. Polymorphicmarkers were screened over a segregating mappingpopulation and mapped. In addition, the following 17SSRs from a map of pear (Yamamoto et al. 2004) wereexamined: NB102a, NB106a, NB111a, NH020a, NH023a,NH029a, NH025a, NB113a, KA4b, BGT23b, HGA8b,NH002b, NH009b, NH004a, NH015a, NH033b, andMSS6 (Yamamoto et al. 2002a–c; Oddou-Muratorio etal. 2001). This SSRs were selected because they map atpositions for which the homologous regions of the applegenome lacked or had only a few SSRs. After verificationthat they generated amplicons in apple, they were mappedin the Fiesta × Discovery population using a range ofannealing temperatures.

Primer design and PCR conditions

Primer pairs flanking the SSR sequence were designedwith the program Primer3 (Rozen and Skaletsky 2000)publicly available at http://fokker.wi.mit.edu/primer3/. Theideal annealing temperature (Tm) of the primers was set at60°C. Some primers were pig-tailed (Brownstein et al.1996), whereby a variable number of nucleotides wasadded to the 5′ end of the reverse primer to obtain thesequence GTTT. Primers were synthesized at Microsynth(Balgach, Switzerland). PCR amplification and tests ofprimers were performed, as described by Gianfranceschi etal. (1998), with the following modifications: the PCR

Table 1 Restriction enzymes, adaptor sequences, and primers used for the construction of the AAG, AAC, and ATC SSR libraries

Library Restr.enzyme

Adaptor (seq1) Adaptor seq 2 (rev complementary of seq1) PCR primer

AAG MboI SAULA: 5′GCGGTACCCGGGAAGCTTGG3′ SAULB: 5′GATCCCAAGCTTCCCGGGTACCGC3′ SAULAAAC MboI SAULA: 5′GCGGTACCCGGGAAGCTTGG3′ SAULB: 5′GATCCCAAGCTTCCCGGGTACCGC3′ SAULAATC Tsp509I TSPAdShort

5′TCGGAATTCTGGACTCAGTGCCAATT3′TSPAdLong5′AATTGGCACTGAGTCCAGAATTCCGA3′

TSPAdShort

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volume was reduced in some cases from 15 to 10 μl;0.07 U/μl reaction of Taq polymerase (New EnglandBioLabs) was used, and the amplification profile wassimplified to an initial denaturation at 94°C for 2 min 30 sfollowed by 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°Cfor 1 min, both for regular (preliminary test of the primers)and with 33P-labeled forward primers. Before loading,radiolabeled PCR products were denatured by the additionof one volume of denaturing gel-loading buffer (Sambrooket al. 1989) and heating at 94°C for 5 min.

SSR alleles were analyzed by running 33P-labeled PCRproducts on a 6% denaturing sequencing gel (NationalDiagnostic, Atlanta, USA) in 1×TBE buffer using a IBI STS-45i DNA sequencing unit. Data scoring was performed, asdescribed by Liebhard et al. (2002), by comparison of theallele size of the nine cultivars with the 33P-labeled sizestandard 30–330 bp (Invitrogen).

Nomenclature for SSR markers

SSR markers developed from enriched genomic librarieswere prefixed with ‘Hi’ (indicating the HiDRAS project,Gianfranceschi and Soglio 2004) followed by a combina-tion of two two-digit numbers, separated by a lower-caseletter. The first two-digit number indicates the number ofthe sequencing plate, the letter and and second two-digitnumber indicate the well of this plate from which the DNAsequence was obtained. SSR markers developed from ESTsequences were designated with the GenBank accessionnumber followed by the subscript “SSR”. Names of theSSRs from the literature were not changed.

Marker quality

The new SSR markers were divided into four qualityclasses based on the type and number of additional,non-SSR containing amplicons that appeared on thegels: (1) clean: no extra band, (2) complementary band(s): all additional bands are complementary; this is at aconstant distance to (specific) SSR bands, (3) extrabands: non-complementary bands are present thatmay hamper multiplexing, (4) dirty: many additional,non-complementary bands that hamper scoring of thetrue SSR bands, not suitable for use in multiplexreactions.

Genetic mapping

The segregation data for the 44 progeny plants genotypedwith the new markers were added to the data previouslyused to develop the “reference maps” of Fiesta andDiscovery (Liebhard et al. 2003b). As these referencemaps were based on 251 progeny plants, missing data wereassigned to the 207 progeny plants not analyzed with thenew SSRs. Mapping of the SSRs was performed withJoinMap™ version 2.0 (Stam and Van Ooijen 1995) in

connection with JMDesk 3.6 provided by Dr. B. Koller(Ecogenics GmbH, Switzerland). A LOD score of 5 wasused to assign markers to linkage groups. Mapping of theSSRs was considered correct (also third-round maps) if theintroduction of the new data did not change, or onlyslightly changed, distances between markers or ordersamong the flanking markers. Drawings of the linkage mapswere generated with MapChart (Voorrips 2001). Some SSRmarkers, which showed no polymorphism in the Fiesta ×Discovery progeny could be mapped in other crosses. Oneof the following three crosses was used in such cases:Discovery × TN10-8, Durello di Forlì × Fiesta, or Fuji ×Mondial Gala, and the map position was estimated bymanual alignment.

Results

One hundred and forty-eight new SSR markers have beendeveloped and mapped, 117 from genomic libraries (65from dinucleotide repeat libraries, 52 from trinucleotiderepeat libraries) and 31 from Malus sequences fromGenBank (24 containing dinucleotide repeats and sevenwith larger repeats). Moreover, it was possible to determinethe location of previously published SSRs of apple (GD147), pear (HGA8b, KA4b, NB102a, NH009b, NH029a,and NH033b) and of a Sorbus torminalis SSR (MSS6). Forall 156 SSRs details of the forward and reverse primers,nucleotides added to the reverse primer to build a pigtail,repeat sequence, repeat type, size-range, number of allelesfor the set of nine cultivars (polymorphism level), mapposition (linkage group), and the quality of the markers ispresented (Table 2). For the EST sequences used to developEST-SSRs, their deduced functions and origins (tissues)have also been indicated (Table 3).

In addition, the allele composition of nine cultivars hasbeen determined to allow the estimation of the level ofpolymorphism of the marker (Table 4). However, in thepresence of only a single allele, it usually remained unclearwhether this was due to the presence of an allele at thehomozygous state or to the presence of one amplified alleleand a null allele (Table 4). These two options could bedistinguished for Fiesta and Discovery based on thesegregation patterns in the mapping population. For othercultivars, this has still to be clarified and so a single valuehas been entered (Table 4). For some SSRs, mainlycontaining trinucleotide repeats, it was not always possibleto distinguish SSR amplicons from other PCR products dueto lack of stutter bands. In such cases, the size of allamplicons is reported in Table 4. This may have led to anoverestimation of the level of polymorphism of these SSRs.

Efficiency of SSR development

The repeat type of the new SSR sequences (2 nt or more)greatly affected the success of development of polymor-phic markers that could be mapped. Sixty-one to sixty-three percent of 2-nt repeats and only 33–40% of the SSRs

205

Tab

le2

Nam

es,p

rimers,pigtailsequence,repeattype,repeatsequence,rang

eandnu

mberof

allelesfoun

din

nine

diploidcultivars,typ

eof

marker,map

positio

nandqu

ality

ofthenewset

ofSSR

markers

developed

SSR

name

Forwardprim

erReverse

prim

erPigtail

seqa

Repeat

type

bRepeat

seq

Allele

rang

eNo.

ofalleles

Type

ofmarkerc

LG

(s)

Qualityd

Hi01a03

CGAATGAAATGTCTA

AACAGGC

AAGCTA

CAGGCTTGTTGATA

ACG

–Perf

AAG

168–19

33

SL

10Clean

Hi01a08

AAGTCCAATCGCACTCACG

CGTA

GCTCTCTCCCGATA

CG

–Com

pAAG-G

A17

7–17

72

SL

16Clean

Hi01b

01GCTA

CAGGCTTGTTGATA

ACGC

ACGAATGAAATGTCTA

AACAGGC

–Perf

AAG

153–18

95

SL

10Com

pl.b

Hi01c04

GCTGCCGTTGACGTTA

GAG

GTTTGTA

GAAGTGGCGTTTGAGG

GTT

Imp

GA

214–23

24

SL

5Com

pl.b

Hi01c06

TTA

GCCCGTA

TTTGGACCAG

GTTTCACCTA

CACACACGCATGG

GTTT

Imp

GT

128–14

43

PML

15Extra

band

sHi01c09

AAAGGCGAGGGATA

AGAAGC

GTTTGCACATTTGAGCTGTCAAGC

GTTT

Perf

GA

214–21

83

SL

14Clean

Hi01c11

TTGGGCCACTTCACAACAG

GTTTGAGTTTGATCTCCAACATTA

CGTTT

Imp

GT

138–26

017

ML

8/16

Clean

Hi01d

01CTGAAATGGAAGGCTTGGAG

GTTTA

CCAATTA

GGACTTA

AAGCTG

GTTT

Com

pGA-G

T19

1–22

25

PML

9Extra

band

sHi01d

05GGTA

TCCTCTTCATCGCCTG

TTA

GATTGACGTTCCGACCC

–Im

pGA

210–>330

16PML

7Extra

band

sHi01d

06GGAGAGTTCCTGGGTTCCAC

AAGTGCACCCACACCCTTA

C–

Imp

GA

115–

165

11ML

11/16

Extra

band

sHi01e10

TGGGCTTGTTTA

GTGTGTCAG

GTTTGGCTA

GTGATGGTGGAGGTG

GTTT

Perf

GA

126–22

48

SL

4Com

pl.b

Hi02a03

GACATGTGGTA

GAACTCATCG

GTTTA

GTGCGATTCATTTCCAAGG

GTTT

Perf

GA

168–19

89

PML

5Extra

band

sHi02a07

TTGAAGCTA

GCATTTGCCTGT

TAGATTGCCCAAAGACTGGG

–Im

pGA

254–31

24

SL

2Extra

band

sHi02a09

ATCTCTA

AGGGCAGGCAGAC

CTGACTCTTTGGGAAGGGC

–Im

pGA

138–15

84

SL

11Clean

Hi02b

07TGTGAGCCTCTCCTA

TTGGG

TGGCAGTCATCTA

ACCTCCC

–Im

pGA

204–21

64

SL

12Clean

Hi02b

10TGTCTCAAGAACACAGCTATCACC

GTTTCTTGGAGGCAGTA

GTGCAG

GTT

Perf

GA

200–25

48

PML

16Clean

Hi02c06

AGCAAGCGGTTGGAGAGA

GTTTGCAACAGGTGGACTTGCTCT

GTTT

Perf

GA

208–25

28

SL

11Clean

Hi02c07

AGAGCTA

CGGGGATCCAAAT

GTTTA

AGCATCCCGATTGAAAGG

GTT

Perf

GA

108–15

05

PML

1Clean

Hi02d

02TTCCTA

GGCTA

CCCGAAATA

TG

GTTTCTGGCATGGACATTCAACC

GTTT

Com

pGA-G

T15

2–19

45

SL

15Clean

Hi02d

04TGCTGAGTTGGCTA

GAAGAGC

GTTTA

AGTTCGCCAACATCGTCTC

GTTT

Perf

GA

224–25

010

SL

10Clean

Hi02d

05GAGGGAGAATCGGTGCATA

GCATCCCTCAGACCCTCATTG

–Perf

GA

153–20

57

SL

12Extra

band

sHi02d

11GCAATGTTGTGGGTGACAAG

GTTTGCAGAATCAAAACCAAGCAAG

GTTT

Imp

GA

198–26

28

SL

14Extra

band

sHi02f06

TAAATA

CGAGTGCCTCGGTG

GCAGTTGAAGCTGGGATTG

GTTT

Perf

GA

204–22

86

SL

15Clean

Hi02f12

ACATGGCCGAAGACAATGAC

GTTTCAACCTTTA

TCCCTCCATCTTTC

GTTT

Perf

GA

130–15

06

SL

17Clean

Hi02g

06AGATA

GGTTTCACCGTCTCAGC

GACCTCTTTGGTGCGTCTG

–Com

pGA-CAC

149–16

34

SL

15Clean

Hi02h

08GCCACTCATA

CCCATCGTA

TTG

GTTTGGCTGGGAATA

TATGATCAGGTG

GTTT

Com

pGT-GA

170–20

06

SL

16Clean

Hi03a03

ACACTTCCGGATTTCTGCTC

GTTTGTTGCTGTTGGATTA

TGCC

GTT

Perf

GA

160–22

810

PML

6Clean

Hi03a06

TGGTGAGAGAAGGTGACAGG

GTTTA

AGGCCGGGATTA

TTA

GTCG

GTTT

Imp

GA

158–19

75

PML

15Dirty

Hi03a10

GGACCTGCTTCCCCTTA

TTC

GTTTCAGGGAACTTGTTTGATGG

GT

Imp

GA

206–29

06

SL

7Clean

Hi03b

03TGAATTGAGTTTGAGAATGGAATG

GTTTGTCAGGACGGGTA

ATCAAGG

GTTT

Perf

GA

196–21

27

SL

12Clean

Hi03c04

CGTA

AATA

GCGAATCCGATA

CC

GTTTCAACATCTGTGGGTCATTGC

GTTT

Perf

GA

169–25

76

SL

10Clean

Hi03c05

GAAGAGAGAGGCCATGATA

CGTTTA

ACTGAAACTTCAATCTA

GG

GTT

Imp

GA

179–22

18

SL

17Clean

Hi03d

06TCATGGATCATTTCGGCTA

AGTTTGCCAATTTTA

TCCAGGTTGC

GTTT

Perf

GA

115–

169

8SL

3Clean

Hi03e03

ACGGGTGAGACTCCTTGTTG

GTTTA

ACAGCGGGAGATCAAGAAC

GTTT

Perf

GA

187–19

96

SL

3Clean

Hi03e04

CTTCACACCGTTTGGACCTC

GTTTCATA

TCCCACCACCACAGAAG

GTTT

Imp

GA

132–16

06

SL

13Clean

Hi03f06

ACGATTTGGTGATCCGATTC

GTTTCGTCGCATTGTGCTTCAC

GTTT

Perf

GA

153–21

79

PML

10Extra

band

sHi03g

06TGCCAATA

CTCCCTCATTTA

CC

GTTTA

AACAGAACTGCACCACATCC

GTTT

Perf

GA

182–20

45

SL

15Clean

Hi04a02

TTCGTGGAAACCTA

ATTGCAG

GTTTCCTCTGCTTCTTCATCTTTGC

GTTT

Perf

GA

82–104

6SL

13Clean

206

SSR

name

Forwardprim

erReverse

prim

erPigtail

seqa

Repeat

type

bRepeat

seq

Allele

rang

eNo.

ofalleles

Type

ofmarkerc

LG

(s)

Qualityd

Hi04a05

GGCAGCAGGGATGTA

TTCTG

GTTTCATGTCAAATCCGATCATCAC

GTTT

Perf

GA

194–22

28

SL

9Clean

Hi04a08

TTGAAGGAGTTTCCGGTTTG

GTTTCACTCTGTGCTGGATTA

TGC

GTT

Perf

GA

211–

250

7SL

5Clean

Hi04b

12CCCAAACTCCCAACAAAGC

GTTTGAGCAGAGGTTGCTGTTGC

GTTT

Perf

GA

140–15

65

SL

8Clean

Hi04c05

AGGATGCTCTGCCTGTCTTC

GTTTCTCACTCGCCTGCTCTA

TCC

GTTT

Perf

GA

179–18

33

SL

15Clean

Hi04c10

TGCGCATTTGATA

GAGAGAGAA

GTTTA

ACAAAGAACGACCCACCTG

GTTT

Perf

GA

172–23

811

ML

3/4

Dirty

Hi04d

02TTCGTGGCTGAGAAAGGAGT

GTTTGTA

CGGTGCATTGTGAAAG

GT

Perf

GA

176–23

815

PML

5Extra

band

sHi04d

10AAATTCCCACTCCTCCCTGT

GTTTGAGACGGATTGGGGTA

GG

Perf

GA

164–18

24

SL

6Extra

band

sHi04e04

GACCACGAAGCGCTGTTAAG

GTTTCGGTA

ATTCCTTCCATCTTG

GTT

Perf

GA

216–24

66

SL

16Clean

Hi04e05

AAGGGTGTTTGCGGAGTTAG

GGTGCGCTGTCTTCCATA

AA

–Perf

GA

144–14

42

SL

8Clean

Hi04f08

CGTGAAAACTCTA

ACTCTCC

GTTTGAAAAGCGCATCAAAGTTCC

GTTT

Perf

GA

218–22

64

SL

10Clean

Hi04f09

ACTGGGTGGCTTGATTTGAG

GTTTCAACTCACACCCTCTA

CATGC

GTTT

Imp

GA

222–26

011

PML

13Com

pl.b

Hi04g

05CTGAAACAGGAAACCAATGC

GTTTCGTA

GAAGCATCGTTGCAG

GTT

Perf

GA

190–25

89

PML

13Clean

Hi04g

11CAGAGGATTA

TCAATTGGACGC

AAACTA

TCTCCAGTTA

TCCTGCTTC

–Perf

GA

118–

164

5SL

11Clean

Hi05b

02GATGCGGTTTGACTTGCTTC

GTTTCTCCAGCTCCCATA

GATTGC

GTTT

Perf

GA

120–17

87

PML

10Clean

Hi05b

09AAACCCAACCCAAAGAGTGG

GTTTCTA

ACGTGCGCCTA

ACGTG

GTTT

Perf

GA

136–14

43

SL

7Clean

Hi05c06

TGCGTGTA

TGGTTGGTTTTG

TGTTTTCTTTGGTTTTA

GTTGGTG

–Com

pGA-G

T13

6–14

25

PML

17Dirty

Hi05d

10AATGGGTGGTTTGGGCTTA

GTTTCTTTGGCTA

TTA

GGCCTGC

GTT

Imp

GT

212–21

22

SL

6Dirty

Hi05e07

CCCAAGTCCCTA

TCCCTCTC

GTTTA

TGGTGATGGTGTGAACGTG

GTTT

Perf

GT

214–23

47

SL

9Extra

band

sHi05f12

TTTGGGTTTGGGTA

GGTTA

GG

GTTTGTGCAGCGCATGCTA

ATG

GTTT

Com

pTA

-CA

157–17

75

ML

12/3

Dirty

Hi05g

12TCTCTA

GCATCCATTGCTTCTG

GTTTGTGTGTTCTCTCATCGGATTC

GTTT

Imp

GT

208–28

810

PML

2Extra

band

sHi06b

06GGTGGGATTGTGGTTA

CTGG

GTTTCATCGTCGGCAAGAACTA

GAG

GTTT

Imp

GT

236–26

24

SL

11Extra

band

sHi06f09

AACCAAGGAACCCACATCAG

GTTTCACTTA

CACACGCACACACG

GTTT

Imp

GT

272–28

84

PML

15Dirty

Hi07b

02ATTTGGGGTTTCAACAATGG

GTTTCGGACATCAAACAAATGTGC

GTTT

Imp

GT

212–21

85

PML

4Clean

Hi07b

06AGCTGCAGGTA

GAGTTCCAAG

GTTTCATTA

CCATTA

CACGTA

CAGC

GTTT

Imp

GT

220–22

64

SL

6Clean

Hi07d

08TGACATGCTTTTA

GAGGTGGAC

GTTTGAGGGGTGTCCGTA

CAAG

GT

Perf

CA

222–23

23

SL

1Extra

band

sHi07d

11CCTTA

GGGCCTTTGTGGTA

AG

GTTTGAGCCGATTA

GGGTTTA

GGG

GTTT

Imp

GT

200–23

411

ML

11/16

Clean

Hi07d

12GGAATGAGGGAGAAGGAAGTG

GTTTCCTCTTCACGTGGGATGTA

CC

GTTT

Imp

GT

184–25

08

ML

2/7

Clean

Hi07e08

TTCGTGCTA

GGGAGTTGTA

GC

GTTTGCCTCCATA

GGATTA

TTTGAC

GTT

Perf

GT

208–24

19

ML

8/3

Clean

Hi07f01

GGAGGGCTTTA

GTTGGGAAC

GTTTGAGCTCCACTTCCAACTCC

GTT

Com

pAT-GT

204–22

05

SL

12Extra

band

sHi07g

10TA

TTGGGTTTTGGGTTTGGA

GTTTCAACCCTTTTGGTTGTGAGG

GTTT

Imp

GT

126–12

83

PML

11Dirty

Hi07h

02CAAATTGGCAACTGGGTCTG

GTTTA

GGTGGAGGTGAAGGGATG

GTT

Perf

GT

246–27

610

SL

17Clean

Hi08a04

TTGTCCTTCTGTGGTTGCAG

GTTTGAAGGTA

AGGGCATTGTGG

GTT

Com

pGAA-G

T24

6–25

44

SL

5Extra

band

sHi08c05

TCATA

TAGCCGACCCCACTTA

GGTTTCACACTCCAAGATTGCATA

CG

GTTT

Perf

AAC

230–24

03

PML

14Extra

band

sHi08d

09AACGGCTTCTTGTCAACACC

GTTTA

CTGCATCCCTTA

CCACCAC

GTTT

Perf

AAC

183–18

62

SL

16Clean

Hi08e04

GCATGGTGGCCTTTCTA

AG

GTTTA

CCCTCTGACTCAACCCAAC

GTTT

Perf

AAG

201–23

46

PML

4Extra

band

sHi08e06

GCAATGGCGTTCTA

GGATTC

GTTTGGCTGCTTGGAGATGTG

GT

Perf

AAC

134–13

84

SL

13Extra

band

sHi08f05

GTGTGGGCGATTCTA

ACTGC

GTTTCCTTTA

TTCTA

AACATGC

CACGTC

GTTT

Perf

AAG

165–16

52

SL

2Clean

Tab

le2(con

tinued)

207

SSR

name

Forwardprim

erReverse

prim

erPigtail

seqa

Repeat

type

bRepeat

seq

Allele

rang

eNo.

ofalleles

Typ

eof

markerc

LG

(s)

Qualityd

Hi08f06

CTTA

GAGCATA

GATGACCTGCAA

GTTTA

GAAATCCAACGGCCAAAG

GTT

Perf

AAG

224–24

25

SL

13Com

pl.b

Hi08f12

GGTTTGTA

ACCCGTCTCTCG

GTTTCGTA

GCTCTCTCCCGATA

CG

GTTT

Com

pAAG-G

A116–22

07

SL

16Extra

band

sHi08g

03ATTCACTTCCACCGCCATA

GGTTTGGAATGATTGCGAGTGAAGC

GTTT

Imp

AAG

104–116

3PML

6Clean

Hi08g

06AATCGAACCAGCACAGGAAG

GTTTA

GATGGAGGTCGTGGTTA

CG

GTTT

Imp

AAG

192–19

82

SL

10Clean

Hi08g

12AGTTCGGTCGGTTCCGTA

AT

GTTTA

GGGCAAGGGGAAAGAAGT

GTTT

Perf

AAG

188–19

74

SL

2Com

pl.b

Hi08h

03GCAATGGCGTTCTA

GGATTC

GGTGGTGAACCCTTA

ATTGG

–Im

pAAC

155–15

82

SL

4Dirty

Hi08h

08TGAACAAATTCCACCACGAA

GTTTGCCAAGGCTA

CAATTTTCA

GTT

Perf

AAC

236–24

23

SL

5Clean

Hi08h

12GAAGGAAATCATCATCAAGACG

GTTTCAAGACCATGGAACAACTTGG

GTTT

Perf

AAG

151–20

36

PML

10Clean

Hi09a01

GAAGCAACCACCAGAAGAGC

GTTTCCCATTCGCTGGTA

CTTGAG

GTTT

Perf

AAG

183–19

27

SL

11Clean

Hi09b

04GCGATGACCAATCTCTGAAAC

TGGGCTTGAATTGGTGAATC

–Im

pAAG

227–27

85

SL

5Clean

Hi09f01

CACCACCAAATTCTCCATCTTC

GTTTA

CCGCCAAATGCTTTGTTA

CGTTT

Imp

AAG

257–26

64

SL

15Clean

Hi11a01

ACCGCCAAATGCTTTGTTA

CGTTTCCTCCATTA

AACTCCTCAGTG

GT

Imp

AAG

214–22

33

SL

15Extra

band

sHi11a03

GGAATTGGAGCTTGATGCAG

GTTTCATA

CGGAATGGCAAATCG

GTTT

Perf

GA

141–14

42

SL

5Com

pl.b

Hi12a02

GCAAGTCGTA

GGGTGAAGCTC

GTTTA

GTA

TGTTCCCTCGGTGACG

GTTT

Imp

AAG

249–25

52

SL

16Clean

Hi12c02

GCAATGGCGTTCTA

GGATTC

GTTTCACCAACAGCTGGGACAAG

GTTT

Imp

AAG

169–19

04

SL

1Extra

band

sHi12f04

ATTGTCCCCACAAAATTGGA

GGGCACACGAGAAAGGATA

A–

Perf

AAC

184–18

72

SL

7Clean

Hi15a13

TTCTCCCCTTCTA

AACCAACC

GGTTTCTTGGCGTA

ACATTG

–Perf

CT

220–23

43

SL

16Clean

Hi15b

02TA

TGGTGGCAACAGTGGAGA

GTTTCGCCACCTCCACTTA

ACATC

GTTT

Imp

GGT

196–20

24

SL

3Com

pl.b

Hi15c07

TCACTTCCCATCATCACTGC

GTTTCAATGTCGAGGCTGGTAATG

GTTT

Perf

ACC

204–21

02

SL

15Clean

Hi15e04

AAACCTCTGCATTCCGTCTC

CTCATA

CTCCTCCCACATTGTC

–Com

pATC-G

AA

209–21

22

SL

5Clean

Hi15g

11TGACATGCATA

GGGTTA

CATGC

GTTTGGGTTCGTA

ATCGTTCTTGTG

GTTT

Perf

ATC

160–16

32

SL

16Clean

Hi15h

12GAACAAGAAGGACGCGAATC

GTTTGGGCCTCGTTA

TCACTA

CCA

GTTT

Perf

ATC

222–22

83

SL

3Clean

Hi16d

02AACCCAACTGCCTCCTTTTC

GTTTCGACATGATCTGCCTTG

GPerf

ATC

144–17

75

SL

11Clean

Hi20b

03AAACTGCAATCCACAACTGC

GTTTA

GTTGCTA

ATGGCGTGTCG

GTT

Imp

AAC

220–24

44

SL

8Clean

Hi21c08

TTCTTCTCCTCCACCACCTC

GTTTGTCACTGAGAAGGCGGTAGC

GTTT

Perf

AAG

227–23

02

SL

5Clean

Hi21e04

TGGAAACCTGTTGTGGGATT

TGCAGAGCGGATGTA

AGTTG

–Im

pAAG

134–16

18

PML

14Clean

Hi21f08

GAGAAAACGCAGAAGCATTG

AGTA

ATGATTTCATCGCGAGTC

–Im

pGAAAA

234–28

210

PML

10Clean

Hi21g

05GACGAGCTCAAGAAGCGAAC

GTTTGCTCTTGCCATTTTCTTTCG

GTTT

Imp

AAG

155–16

43

ML

1/15

Clean

Hi22a07

CTCTTCCTTCTCCGCCTCTT

GTTTCACTCAGAATGCCTCACAGC

GTTT

Imp

AAG

153–20

27

PML

5/10

Clean

Hi22d

06CCCCGAGCTCTA

CCTCAAA

CATTA

TGTTTCCGGTTTTTGG

–Im

pAAC

126–13

54

SL

2Clean

Hi22f04

TCAATCCTCTGCTCTTCAAGG

GTTTA

ATCACCTGCTGCTGCTTG

GTTT

Perf

AAG

135–14

74

SL

10Clean

Hi22f06

CAATGGCGTCTGTGTCACTC

GTTTA

CGACGGGTA

AGGTGATGTC

GTTT

Perf

AAG

240–24

63

SL

16Clean

Hi22f12

GGCCTCACCCAGTCTACATT

GTTTGGTGTGATGGGGTA

CTTTGC

GTTT

Imp

AAG

205–21

74

SL

5Clean

Hi22g

06GAAAGGACCGAATTTCATGC

GTTTGTCTA

CAACTGTCTGATGGTA

GC

GTT

Imp

AAG

240–24

92

SL

8Extra

band

sHi23b

12TGAGCGCAATGACGTTTTA

GGTTTCAGGCTTTCCCTTCAGTGTC

GTT

Perf

AAC

142–16

93

SL

14Extra

band

sHi23d

02CCGGCATA

TCAAAGTCTTCC

GTTTGATGGTCTGAGGCAATGGAG

GTTT

Imp

AAG

157–16

63

SL

11Extra

band

sHi23d

06TTGAAACCCGTA

CATTCAACTC

GTTTCAAGAACCGTGCGAAATG

GTT

Perf

AAC

158–18

85

SL

9Com

pl.b

Hi23d

11b

GACAGCCAGAAGAACCCAAC

GTTTA

TTGGTCCATTTCCCAGGAG

GTTT

Perf

CTT

177–18

44

PML

4Clean

Tab

le2(con

tinued)

208

SSR

name

Forwardprim

erReverse

prim

erPigtail

seqa

Repeat

type

bRepeat

seq

Allele

rang

eNo.

ofalleles

Type

ofmarkerc

LG

(s)

Qualityd

Hi23g

02TTTTCCAGGATA

TACTA

CCCTTCC

GTTTCTTCGAGGTCAGGGTTTG

GT

Perf

AAC

230–25

76

SL

4Com

pl.b

Hi23g

08AGCCGTTTCCCTCCGTTT

GTTTGTGGATGAGAAGCACAGTCA

GTT

Perf

CTT

211–

220

3SL

4Clean

Hi23g

12CCCTTCCCTA

CCAAATGGAC

GTTTA

AAGGGGCCCACAAAGTG

GTTT

Com

pCAA-A

GC-

CCA

223–24

14

PML

8Clean

Hi24f04

CCGACGGCTCAAAGACAAC

TGAAAAGTGAAGGGAATGGAAG

–Im

pAAG

144–15

35

SL

2Clean

AF05

7134

SSR

ACTA

CCCAATGCCCACAAAG

TATCCTCGCCCAAAAGACTG

–Perf

GA

202–22

47

SL

10Com

pl.b

AF52

7800

SSR

TGGAAAGGGTTGATTGACCT

AACAGCGGGTGGTA

AATCTC

–Perf

GA

168–19

45

SL

17Com

pl.b

AJ000761a

SSRe

CTGGGTGGATGCTTTGACTT

TCAATGACATTA

ATTCAACTTA

CAAAA

–Im

pTA

260–27

25

SL

14Clean

AJ000

761b

SSR

CCCTA

AACACACAGCCTCCT

GTTTCAGCATCGCAGAGAACTGAG

GTTT

Perf

GA

210–20

88

SL

14Clean

AJ001

681 S

SR

CCTGAGGTTA

TTGACCCAAAA

CACTCAGTTGGAAAACCCTA

CA

–Perf

GA

169–19

56

SL

17Clean

AJ251116 S

SR

GATCAGAAAATTGCTA

GGAAAAGG

AGAGAACGGTGAGCTCCTGA

–Perf

GA

165–16

72

SL

2Com

pl.b

AJ320

188 S

SR

AACGATGCTTGAGGAAGAACA

GCTTA

ACAGAAACATCGCTGA

–Perf

GA

191–24

58

SL

9Clean

AT00

0174

SSR

CGGAGGCCGCTA

TAATTA

GG

CCTGGAAAGAAAGTA

AAAGGACA

–Com

pTA

-GTA

-GT

178–20

06

PML

17Extra

band

sAT00

0400

SSR

CTCCCTTTGCTCCCTCTCTT

AGGATGTCAGGGTTGTA

CGG

–Im

pCAG

198–23

27

SL

2Extra

band

sAT00

0420

SSR

TTGGACCAATTA

TCTCTGCTA

TT

GATGTGGTCAGGGAGAGGAG

–Im

pGA

189–20

95

SL

4Extra

band

sAU22

3486

SSR

TGACTCCATGGTTTCAGACG

AGCAATTCCTCCTCCTCCTC

–Com

pGAA-G

A20

5–21

73

SL

13Extra

band

sAU22

3548

SSR

ACCACCACTGCAGAGACTCA

GACGCACCCATTCATCTTTT

–Perf

GGA

262–27

84

SL

10Extra

band

sAU22

3657

SSR

TTCTCCGTCCCCTTCAACTA

CACCTTGAGGCCTCTGTA

GC

–Im

pGA

219–23

36

SL

3Clean

AU22

3670

SSR

GGACTCAATGCCTTTTCTGG

AGGATGGCAGCAATCTTGAA

–Perf

ACC

194–20

23

PML

5Extra

band

sAU30

1431

SSR

TCTTCCTCCTCCTCCTCCTC

TCTTTTTCTTGGGGTCTTGG

–Perf

AAG

213–21

62

SL

16Extra

band

sAY18

7627

SSR

GAGGACTGAATTGGTTGAGGTC

GTTTCTCACCCGTA

TATA

GGCCAAC

GTTT

Com

pGT-TA

300–30

02

SL

17Clean

CN44

4542

SSR

ATA

AGCCAGGCCACCAAATC

GTTTGCAGTGGATTGATGTTCC

GT

Perf

GA

110–

156

8PML

9Clean

CN44

4636

SSR

CACCACTTGAGTA

ATCGTA

AGAGC

GTTTGCCAGTTA

AGGACCACAAGG

GTTT

Com

pAT-GT

239–24

33

SL

2Clean

CN44

4794

SSR

CATGGCAGGTGCTA

AACTTG

GTTTGCAACTCACACAATGCAAC

GTT

Perf

TA23

0–30

68

PML

7Extra

band

sCN44

5290

SSR

TCTCAGTTGCTCTGGCTTTG

GTTTGCAATCAATGCCACTCTTC

GTT

Perf

GA

230–24

23

SL

6Clean

CN44

5599

SSR

TCAAATGGGTTCGATCTTCAC

GTTTGCCTGGCTGTA

ACTGTTTGG

GTTT

Perf

TA13

0–17

611

PML

5Extra

band

sCN49

1050

SSR

CGCTGATGCGATA

ATCAATG

GTTTCACCCACAGAATCACCAGA

GTT

Perf

GA

>33

0–>33

03

SL

11Clean

CN49

3139

SSR

CACGACCTCCAAACCTA

TGC

GTTTA

TGAAAGTA

CGGCACCCATC

GTTT

Perf

TA12

4–16

210

PML

2Clean

CN49

6002

SSR

TCAGAATCTCAAGCAAGATCCTC

GTTTGATTGATCGTGGCGATA

TG

GTT

Com

pTA

-CTT

243–26

14

SL

5Clean

CN49

6913

SSR

TGCCTTTGAGAATCGAAATG

TGTTTGTCAATTTCTTGGAACTC

–Perf

TA23

6–27

85

SL

12Clean

CN58

1493

SSR

GCTTTTCATGGTGGAAAAACTG

GTTTGACTCTCCGCTCTGATGGAC

GTTT

Perf

TA18

4–22

88

SL

2Clean

U78

948 S

SR

GATCGTCCGCCACCTTA

AT

AGGGTTTTCATCATGCACATT

–Im

pTA

178–19

07

SL

14Extra

band

sU78

949 S

SR

TTTGTCTA

CCTCTGATCTTA

ACCAA

CAGCATCGCAGAGAACTGAG

–Perf

GA

172–22

512

ML

6/14

Extra

band

sZ38

126 S

SR

AAGAGGGTGTTCCCAGATCC

TGTTCGATGTGACTTCAATGC

–Perf

TA21

4–24

03

SL

7Clean

Z71

980 S

SR

TCTTTCTCTGAAGCTCTCATCTTTC

GGACATGGATGAAGAATTGGA

–Im

pTA

170–17

22

SL

8Clean

Z71

981 S

SR

GCACTTA

CCTTTGTTGGGTCA

CCGGCATTCCAAATGTA

ACT

–Perf

AAG

212–23

25

SL

15Clean

GD14

7TCCCGCCATTTCTCTGC

GTTTA

AACCGCTGCTGCTGAAC

GTTT

uGA

135–15

56

SL

13Dirty

Tab

le2(con

tinued)

209

with 3-nt repeats (or more) could be mapped (Table 5). Theorigin of the sequence (enriched library or GenBankaccession) did not affect the success rate (Table 5). Therepeat type similarly affected the observed allelic diversityof the markers, being highest for the 2-nt repeats (Table 5).However, if the average number of repeats found in thesequence used for the development of the SSRs isconsidered, it can be affirmed that the higher allelicvariation observed for the 2-nt containing SSRs is probablydue to the higher number of repeats. In fact, sequencesfrom 2-nt repeats genomic libraries and ESTs contain onaverage 28 and 14 repeats, respectively, while 3-nt repeatgenomic libraries and ESTs contain nine and six repeats,respectively (Table 5).

A relatively high number of SSRs (41%) developed forpear and Sorbus torminalis were transferred to the apple.Surprisingly, only one out of eight previously publishedapple SSRs (GD series) could be mapped (Table 5).

To be efficiently used in genotyping projects, SSRs needto be sufficiently polymorphic and easy to score. If thethreshold to declare a single-locus SSR as sufficientlypolymorphic within our current set of nine cultivars is set atfive alleles, 62% of the 2-nt repeat SSRs and 24% ≥3-ntrepeat SSRs can be considered (Table 6). The two mostpolymorphic single-locus SSRs were Hi02d04 andHi07h02, both being 2-nt repeat SSR with ten alleles inthe set of nine cultivars.

Approximately 58 and 10% of the new SSRs have beenclassified as being “clean” or showing “complementarybands” (amplification of amplicons at a constant distancefrom the SSR allele), respectively. On the other hand, 26and 6% of the new markers have been classified as “extrabands” or “dirty”, respectively. Under our PCR conditions,these SSRs amplify several additional non-SSR amplifica-tion products, which make them unsuitable for high-throughput genotyping. The SSR amplicons of the markersclassified as “extra bands” are clearly visible, while thoseof class “dirty”may be difficult to recognize. Improvementof these SSRs by the design and testing of alternativeprimers was not pursued unless they are shown to belocated in regions of high interest in which no other, highquality SSRs are located.

Genetic mapping

One hundred and forty-eight SSRs out of 156 have beenmapped on the reference map derived from the crossFiesta × Discovery (Liebhard et al. 2003b). The remainingeight were mapped in other crosses: four in Discovery ×TN10-8 (Hi23b12, Hi01c09, Hi09f01, Hi08f05), three inDurello di Forlì × Fiesta (Hi03c05, Hi08d09, Z71980SSR)and one in Fuji × Mondial Gala (Hi11a01) (Fig. 1).

The 156 new SSR primer pairs enriched the referencemap with 168 new loci (12 primer pairs amplified two locithat could both be mapped). The linkage groups (LGs) withhighest increase in loci are LG 16 with 16 loci and LGs 10and 5 with 14 loci each, followed by LGs 2, 11, and 15 with13 loci each. The LG with the lowest increase of loci is LGS

SR

name

Forwardprim

erReverse

prim

erPigtail

seqa

Repeat

type

bRepeat

seq

Allele

rang

eNo.

ofalleles

Type

ofmarkerc

LG

(s)

Qualityd

HGA8b

AACAAGCAAAGGCAGAACAA

CATA

GAGAAAGCAAAGCAAA

(Tm

55°C

)–

Com

pGA-G

CTTT

133–16

46

ML

3/11

Dirty

KA4b

AAAGGTCTCTCTCACTGTCT

CCTCAGCCCAACTCAAAGCC

(Tm

55°C

)–

Imp

GT

137–14

13

SL

1Com

pl.b

NB10

2aTGTTA

TCACCTGAGCTA

CTGCC

CTTCCTCTTTA

TTTGCCGTCTT

–Perf

GA

181–18

33

PML

16Extra

band

sNH00

9bCCGAGCACTA

CCATTGA

CGTCTGTTTA

CCGCTTCT

–Perf

GA

138–16

26

SL

13Extra

band

sNH02

9aGAAGAAAACCAGAGCAGGGCA

CCTCCCGTCTCCCACCATA

TTA

G(Tm

55°C

)–

Perf

GA

91–99

5SL

9Com

pl.b

NH03

3bGTCTGAAACAAAAAGCATCGCAA

CTGCCTCGTCTTCCTCCTTA

TCTCC

–Perf

GA

163–18

97

SL

2Clean

MSS6

CGAAACTCAAAAACGAAATCAA

ACGGGAGAGAAACTCAAGACC

–u

GT

273–27

93

SL

4Extra

band

sa N

ucleotides

addedto

theprim

erto

create

apigtail(Brownstein

etal.19

96)

bPerfect

(Perf),im

perfect(Imp),compo

und(Com

p)(W

eber

1990)

c Singlelocus(SL):foreach

sample,amaxim

umof

only

twoallelesisam

plifiedandthemarkerhasbeen

mappedto

asing

lelocus;multilocus

(ML):morethan

twoallelesforeach

sample

isam

plifiedandtheallelesam

plifiedwith

themarkerhave

been

mappedto

twodifferentloci;presum

edmultilocus

(PML):morethan

thetwoallelespresentor

presence

ofotherband

ssimilarto

SSR

alleles,thesecond

locuswas

notmapped(not

polymorph

),dClean:n

oextraband

s;complem

entary

band

(s)(Com

pl.b

.):the

only

extraband

(s)visibleareataconstant

distance

from

theSSRallele;E

xtra

band

s:theSSRallelesareclearlyvisiblebu

totheram

plicons(few

ormany)

arepresent;multip

lexing

may

bedifficult;Dirty:manynonSSR-likeam

pliconsvisible,

scoringof

theSSR

difficult;multip

lexing

notadvised

e For

thisEST,

twopairsof

prim

ershave

been

developed,

AJ000

761a

andb

Tab

le2(con

tinued)

210

1 with only five new loci. The largest distances betweentwo flanking SSRs are on Discovery 6 (36.2 cM) and Fiesta7 (37.5 cM). The maps of Fiesta and Discovery have beenenriched by 99 and 115 loci, respectively (54 loci incommon). The maps now span a total of 1,145.3 cM(Fiesta) and 1,417.1 cM (Discovery). This corresponds toan increase of 1.5 cM for the map of Fiesta and a decreaseof 37.5 cM for the map of Discovery compared with themaps of Liebhard et al. (2003b). Most of the reduction ofthe total length of the map of Discovery is due to thesplitting of its LG3 (reduction of 10 cM). Also, the averagechromosome lengths of Fiesta and Discovery did notchange substantially, being 67.4 cM (previously 67.4 cM)and 83.35 cM (previously 85.6 cM), respectively. The factthat no substantial changes in the total length of the

parental maps are observed is an indication that the genomecoverage is close to completion.

Selection of a genome covering set of apple SSRs

To facilitate efficient genome-wide mapping approaches,we aimed at developing a set of 100 SSRs that cover theentire apple genome. The high number of mapped SSRs inFiesta × Discovery as well as in various other availablemapping populations (data not shown) allowed the firstdesign of such a set. Eighty-six SSRs were selected thatspan around 85% of the apple genome and that have anaverage distance between markers of 15 cM (Fig. 2).Regrettably, SSRs for 16 regions are not yet available. Out

Table 3 Description and putative functions of the mapped ESTs as found at NCBI GenBank

SSR namea Definition

AF057134-SSR Malus domestica NADP-dependent sorbitol 6-phosphate dehydrogenase (S6PDH) gene, complete cdsAF527800-SSR Malus x domestica expansin 3 (EXP03) mRNA, complete cdsAJ000761a,b-SSR Malus domestica mRNA for MADS-box protein, MADS7AJ001681-SSR Malus domestica mRNA for MADS box protein MdMADS8AJ251116-SSR Malus domestica mRNA for B-type MADS box protein (mads13 gene)AJ320188-SSR Malus domestica mRNA for MADS box protein (MADS12A gene)AT000174-SSR AT000174 Apple young fruit cDNA library Malus x domestica cDNA clone af180, mRNA sequenceAT000400-SSR AT000400 Apple peel cDNA library Malus x domestica cDNA clone ap189, mRNA sequenceAT000420-SSR AT000420 Apple peel cDNA library Malus x domestica cDNA clone ap212, mRNA sequenceAU223486-SSR AU223486 Apple shoot cDNA library Malus x domestica cDNA clone S0016, mRNA sequenceAU223548-SSR AU223548 Apple shoot cDNA library Malus x domestica cDNA clone S0279, mRNA sequenceAU223657-SSR AU223657 Apple shoot cDNA library Malus x domestica cDNA clone S0159, mRNA sequenceAU223670-SSR AU223670 Apple shoot cDNA library Malus x domestica cDNA clone S0086, mRNA sequenceAU301431-SSR AU301431 Apple shoot cDNA library Malus x domestica cDNA clone S1069, mRNA sequenceAY187627-SSR Malus x domestica S-RNase (S) gene, S9 allele, partial cdsCN444542-SSR Mdfw2003g22.x1 Mdfw Malus x domestica cDNA clone Mdfw2003g22 3-similar to TR:Q9SSL1 Q9SSL1 F15H11.6

Hypothetical protein At1g70810CN444636-SSR Mdfw2003l01.x1 Mdfw Malus x domestica cDNA clone Mdfw2003l01 3-similar to TR:O81077 O81077 PUTATIVE

CYTOCHROME P450, mRNA sequenceCN444794-SSR Mdfw2001i05.y1 Mdfw Malus x domestica cDNA clone Mdfw2001i05 5-, mRNA sequenceCN445290-SSR Mdfw2002h21.y1 Mdfw Malus x domestica cDNA clone Mdfw2002h21 5-, mRNA sequenceCN445599-SSR Mdfw2003f11.y1 Mdfw Malus x domestica cDNA clone Mdfw2003f11 5-similar to TR:O81062 O81062 T18E12.21

Hypothetical protein At2g03120CN491050-SSR Mdfw2008p11.y1 Mdfw Malus x domestica cDNA clone Mdfw2008p11 5-, mRNA sequenceCN493139-SSR Mdfw2012f06.y1 Mdfw Malus x domestica cDNA clone Mdfw2012f06 5-similar to TR:O81808 O81808

HYPOTHETICAL 62.6 KD PROTEIN, mRNA sequenceCN496002-SSR Mdfw2021d09.y1 Mdfw Malus x domestica cDNA clone Mdfw2021d09 5-similar to TR:O23131 O23131

HYPOTHETICAL 37.1 KD PROTEIN, mRNA sequenceCN496913-SSR Mdfw2023a24.y1 Mdfw Malus x domestica cDNA clone Mdfw2023a24 5-, mRNA sequenceCN581493-SSR Mdfw2039o14.y1 MdfwMalus x domestica cDNA clone Mdfw2039o14 5-similar to TR:Q9ZQF5 Q9ZQF5 PUTATIVE

RING-H2 FINGER PROTEIN, mRNA sequenceU78948-SSR Malus domestica MADS-box protein 2 mRNA, complete cdsU78949-SSR Malus domestica MADS-box protein 3 mRNA, complete cdsZ38126-SSR M. domestica gene for calmodulin-binding protein kinaseZ71980-SSR M. domestica mRNA for knotted1-like homeobox proteinZ71981-SSR M. domestica partial gene for kn1-like proteinaThe first part of the SSR name corresponds to sequence accession number

211

Tab

le4

Allele

compo

sitio

nof

nine

diploidcultivars

SSR

name

Fiesta

Discovery

Florina

Nov

aEasaygro

TN10

-8Durello

diForlì

Prima

Mon

dial

Gala

Fuji

Hi01a03

168:19

317

8:19

316

8nd

193nd

193nd

nd19

3nd

Hi01a08

null

177:nu

llnu

llnu

llnu

ll17

7nu

ll17

7nu

llHi01b

0116

5:18

917

4:18

916

516

215

315

318

916

218

915

316

218

9nd

Hi01c04

218:22

222

2:23

221

821

423

221

421

821

421

822

221

8nd

ndHi01c06

142:14

212

8:14

2nd

128

142

12814

214

414

214

2Hi01c09

216:21

821

6:21

821

621

821

621

621

421

621

621

621

821

621

8Hi01c11

216:22

0x26

0:26

0a

13820

0nu

ll:nu

lla23

4:24

0y

13820

413

815

220

220

622

013

814

615

220

622

013

814

414

620

423

424

013

814

820

020

424

013

814

817

622

013814415221613

814

815

221

8Hi01d

0119

9:22

019

5:22

019

922

019

519

119

519

522

219

119

519

9nd

Hi01d

0532

0:>33

0a

21024

232

0:nu

ll21

022

224

430

0>33

0b

21022

2>33

0a/b

21222

624

2>33

0a/b

210>33

0b

21024

231

821

022

232

8212222242300

320>330c/d

21024

232

2>33

0a/b

Hi01d

0612

5:13

312

4:13

115

516

313

516

513

713

713

713

112

411515

512

915

5Hi01e10

220:22

021

4:12

622

022

422

420

420

818

220

820

421

222

4nd

Hi02a03

176:18

617

8:18

617

017

616

817

019

818

418

817

617

418

617

618

6Hi02a07

254:28

228

0:28

031

231

225

431

228

028

028

028

2Hi02a09

138

138:15

815

813

813

815

813

815

213

814

8nd

Hi02b

0721

4:nu

llnu

ll21

621

620

421

6nd

20421

620

4nd

Hi02b

1022

8:22

8a20

2:20

2a22

6:nu

ll20

0:20

0a20

022

620

022

420

022

622

624

020

020

222

622

422

625

420

222

422

6Hi02c06

230:24

423

0:24

225

220

824

224

225

223

222

724

221

224

2nd

Hi02c07

116:15

015

0:15

0116118

114118

116118

116

108118

116

ndHi02d

02nu

ll:nu

ll15

2:nu

llnd

15419

415

619

415

219

4nd

ndHi02d

0421

8:25

022

6:24

623

423

024

023

425

022

423

224

024

422

4nd

Hi02d

0515

3:19

715

3:20

515

320

515

317

515

317

515

317

315

317

515

319

119

520

5Hi02d

1123

4:24

424

4:19

823

425

825

425

826

224

419

825

419

824

625

824

826

2Hi02f06

216:22

420

8:21

421

622

820

821

420

422

821

421

620

822

821

622

8nd

Hi02f12

130:13

214

0:14

013

415

013

213

015

013

815

0nd

13215

0nd

Hi02g

0616

1:16

116

1:16

316

116

116

116

314

915

516

116

116

316

116

116

3Hi02h

0817

2:17

217

0:17

817

217

217

217

420

017

218

017

217

2Hi03a03

188:19

222

417

2:19

622

422

816

022

816

022

422

816

017

222

818

619

421

822

818

619

421

822

8nd

nd

Hi03a06

158:16

017

819

715

8:19

716

017

819

716

017

819

715

816

019

7nd

16017

818

319

715

819

7nd

Hi03a10

216:24

021

6:28

421

624

021

629

0nd

20622

6nd

ndnd

Hi03b

0321

0:21

220

4:21

020

621

020

020

620

021

019

620

820

021

021

221

0Hi03c04

169:nu

llnu

ll16

920

120

117

520

125

720

120

116

920

1Hi03c05

205:22

120

5:22

117

919

519

119

321

520

9nd

ndnd

Hi03d

06117:117

143:16

913

314

314

311714

511514

7117

117

ndHi03e03

193:19

919

3:19

719

918

919

919

319

918

919

818

719

919

9nd

Hi03e04

148:16

013

2:14

813

213

215

014

614

616

013

416

015

016

013

2

212

SSR

name

Fiesta

Discovery

Florina

Nov

aEasaygro

TN10

-8Durello

diForlì

Prima

Mon

dial

Gala

Fuji

Hi03f06

153:17

715

3:17

915

316

117

915

3211

15321

715

317

620

521115

3211

15317

721

2nd

Hi03g

0619

8:20

018

2:19

818

219

618

219

618

2nd

204

19620

419

6Hi04a02

82:102

90:104

102

102

8810

010

288

102

8210

210

2Hi04a05

194:20

419

4:19

819

420

420

422

219

820

819

420

221

022

219

420

421

6nd

ndHi04a08

226:24

621

6:25

021

621

821

221

621

221

621

221

621

221

621

622

6nd

Hi04b

1215

0:15

614

0:15

615

614

215

615

015

614

615

015

614

215

614

215

6Hi04c05

181:18

118

3:18

118

118

118

118

118

317

918

317

918

318

1Hi04c10

230:nu

llx20

6:20

6y

176

228:23

8x20

0:20

6y20

623

420

623

420

423

417

217

220

223

417

820

223

417

220

223

4

Hi04d

0220

8:22

223

8:nu

ll18

019

417

618

220

418

219

020

419

019

822

218

620

421

421

819

020

421

417

619

222

218

620

421

822

2Hi04d

1016

4:16

4nu

ll16

418

218

016

416

418

2nd

16418

0Hi04e04

226:24

622

2:22

822

824

621

624

621

622

223

022

624

622

824

621

622

6Hi04e05

144:nu

llnu

llnu

llnu

llnd

null

null

144

null

Hi04f08

202:21

821

8:22

622

622

622

021

822

022

622

621

8Hi04f09

22224

425

423

0b22

424

422

222

425

426

022

422

625

225

822

425

622

4;25

422

422

624

426

0222224244254nd

Hi04g

05nu

ll:nu

ll23

019

0:19

423

019

425

823

225

824

025

423

023

023

023

224

8Hi04g

11nu

ll:nu

ll16

6118:nu

ll118

158

158

140

158

164

118

Hi05b

0212

6:12

612

0:12

212

617

812

217

813

416

212

014

416

217

812

212

612

617

812

613

4Hi05b

0914

0:14

413

6:13

613

613

614

413

614

013

614

014

4nd

ndHi05c06

139:13

813

6:13

613

814

113

813

814

114

114

213

813

813

913

613

8Hi05d

1021

2:nu

llnu

ll21

2nd

212

212

nd21

221

2Hi05e07

212:21

421

6:23

021

423

0nu

ll21

423

4nu

ll21

423

021

422

8Hi05f12

173:

nullx16

9:16

9y17

3:17

7X15

7:16

9y15

717

315

717

115

717

315

717

315

717

315

717

317

715

717

3Hi05g

1223

0b20

821

623

821

621

623

821

6.19

221

623

822

224

220

821

623

023

820821622022821

6

Hi06b

0624

2:26

223

6:26

226

026

026

023

626

026

026

224

226

0Hi06f09

280:28

827

2:27

428

028

827

228

827

228

827

428

828

827

228

027

228

8Hi07b

0221

4:21

821

4:nu

ll21

221

621

421

6nd

212

nd21

621

6Hi07b

0621

0:22

022

0:22

222

622

622

622

622

222

622

222

622

222

6Hi07d

0822

2:23

222

2:22

6nd

ndnd

ndnd

ndnd

Hi07d

1121

4:21

8x22

2:23

4y21

6:22

6x23

4:nu

lly

200:20

021

422

022

021

623

222

820

021

822

620

022

021

222

0

Hi07d

1224

6:nu

llx18

4:nu

lly

194:21

8x24

625

024

824

819

824

619

824

618

420

625

0Hi07e08

208:20

8x23

3:23

5y20

8:21

2x23

1:24

1y20

822

223

123

320

823

320

823

323

420

823

323

421

022

223

1nd

nd

Hi07f01

204:21

420

4:21

020

622

021

021

021

022

021

022

021

020

621

0Hi07g

1012

6:nu

ll12

8.nu

llnd

ndnd

ndnd

ndnd

Tab

le4(con

tinued)

213

SSR

name

Fiesta

Discovery

Florina

Nov

aEasaygro

TN10

-8Durello

diForlì

Prima

Mon

dial

Gala

Fuji

Hi07h

0225

6:26

427

0:27

624

625

627

625

626

826

624

827

424

826

424

626

0Hi08a04

246:25

424

6:25

024

624

824

624

825

024

625

424

625

024

625

424

625

4Hi08c05

231:23

123

1:24

023

124

023

123

424

023

124

023

123

423

123

423

123

424

023

123

424

0Hi08d

0918

318

318

618

318

318

318

6nd

nd18

318

6Hi08e04

207:21

620

1:nu

ll21

620

720

121

621

622

521

620

723

420

7Hi08e06

134:13

813

4:nu

ll13

413

413

413

413

713

413

413

813

4Hi08f05

165

165

165

nd16

515

716

5nd

ndnd

Hi08f06

227:23

022

4:24

223

023

022

422

723

023

322

723

022

723

023

0Hi08f12

131:21

8116:13

111622

011621

820

613

120

615

821

821

822

012

922

0Hi08g

03113:11610

4116:11610

410

4113116

ndnd

104113

104113

104113116

104113116

Hi08g

0619

2:19

819

2:19

219

219

819

219

819

219

219

219

8nd

192

Hi08g

1218

8:18

818

8:19

418

819

419

119

418

819

418

819

718

818

819

118

819

1Hi08h

0315

5:15

515

5:15

815

515

515

5nd

nd15

5nd

Hi08h

0823

6:23

623

6:23

923

624

223

623

624

223

623

923

623

923

623

923

6Hi08h

1215

7:20

316

3:16

916

920

315

120

315

716

315

116

920

315

117

215

717

2Hi09a01

183,18

618

6:19

218

318

618

0,18

318

3,18

618

3,19

218

3,18

618

618

3,19

2Hi09b

0423

0:22

727

8:27

824

222

726

922

724

222

724

223

024

224

226

923

024

2Hi09f01

257:26

025

7:26

626

026

025

726

025

726

625

726

626

026

0Hi11a01

214:21

721

4:21

721

721

721

721

421

721

422

321

422

321

7Hi11a03

141:14

114

1:14

414

114

114

114

414

114

414

114

114

1Hi12a02

249:25

525

5:25

524

925

524

925

524

925

524

925

525

525

525

5Hi12c02

190:17

816

9:nu

ll17

816

917

816

917

816

916

919

017

8Hi12f04

184:18

418

4:18

718

718

418

418

418

418

418

718

4Hi15a13

220:23

222

0:22

023

223

423

222

022

023

223

2Hi15b

0219

6:19

919

9:19

920

2nu

ll20

220

219

919

920

2Hi15c07

210:21

020

4:21

020

421

021

021

020

421

021

020

421

020

421

0Hi15e04

209:20

920

9:21

220

920

921

220

920

920

921

220

920

921

2Hi15g

1116

0:16

316

0:16

016

316

3nd

160

16016

316

016

316

3Hi15h

1222

2:22

222

2:22

522

222

222

222

222

822

222

522

222

522

2Hi16d

0214

4:14

414

4:14

714

416

514

414

416

514

417

714

414

414

415

3Hi20b

0322

0:23

822

0:22

9nd

ndnd

22024

422

922

024

4nd

Hi21c08

230:23

023

0:22

723

022

723

022

723

022

723

022

723

023

022

7nd

Hi21e04

134:13

414

915

615

816

113

4:13

614

915

615

816

113

413

614

915

813

614

915

615

816

113

414

915

816

113

413

814

814

915

816

113

413

614

915

816

113

613

814

915

616

113

413

814

915

616

1Hi21f08

242:24

628

028

224

2:24

223

424

425

027

228

224

224

828

028

224

224

826

628

024

225

028

024

225

028

225

026

627

228

024

424

827

228

028

224

424

828

2

Hi21g

0515

8:16

415

5:15

8:16

415

515

815

515

515

816

415

815

515

816

415

515

816

415

515

8Hi22a07

192:19

8x15

3:18

9y:

196:19

6anu

ll:nu

lla19

220

215

319

620

215

318

920

215

320

218

919

219

618

919

820

218

919

8Hi22d

0612

9:12

912

6:12

913

212

913

512

913

513

212

612

912

913

512

913

5

Tab

le4(con

tinued)

214

SSR

name

Fiesta

Discovery

Florina

Nov

aEasaygro

TN10

-8Durello

diForlì

Prima

Mon

dial

Gala

Fuji

Hi22f04

138:14

713

5:13

814

114

714

113

813

813

814

113

814

113

8Hi22f06

240:24

624

3:24

624

024

624

024

624

624

324

624

024

624

024

6Hi22f12

211:nu

ll211:21

721

421

720

521

721121

720

5211

217

Hi22g

0624

0:24

924

0:24

024

024

024

024

024

024

924

0Hi23b

1215

4:16

915

4:16

915

416

914

216

914

216

916

914

215

415

416

914

2Hi23d

0216

0:16

616

0:16

015

716

016

015

716

016

016

016

015

716

0Hi23d

0616

1:16

115

8:16

117

017

316

117

016

116

116

116

117

016

118

815

818

8Hi23d

11b

181:18

417

717

8:18

417

717

718

417

718

417

717

818

417

717

818

117

718

417

717

818

417

718

4Hi23g

0224

8:25

725

4:25

723

923

925

523

924

825

523

925

523

024

8Hi23g

0822

0:22

021

4:22

0211

21122

022

021122

021122

021

422

022

021122

0Hi23g

1222

3:22

324

122

3:22

624

122

624

122

623

524

122

322

623

524

122

322

623

524

122

322

623

524

122

323

524

122

323

524

1

Hi24f04

144:15

014

7:15

315

315

315

015

315

315

015

314

415

015

215

3AF05

7134

SSR

210:21

620

8:22

420

821

621

621

422

021

620

821

620

221

6AF52

7800

SSR

178:nu

ll16

8:18

416

816

816

816

816

819

416

817

8nd

AJ000

761a

SSR

262:26

226

2:26

626

226

426

026

226

226

426

026

626

427

2nd

ndAJ000

761b

SSR24

8:24

821

0:24

824

424

821

025

224

624

820

822

621

025

022

624

424

424

8AJ001

681 S

SR

169:19

118

9:19

116

918

716

918

718

318

919

519

116

918

9AJ251116 S

SR

165:16

716

7:16

716

716

716

716

5nd

nd16

7nd

AJ320

188 S

SR

213:nu

llnu

ll:nu

ll19

919

921

320

320

721

919

124

519

919

919

9AT00

0174

SSR

186:19

418

6:19

217

818

617

818

618

618

817

8nd

17820

018

819

417

8AT00

0400

SSR

198:21

622

621

6:21

621

022

423

221

022

423

219

821

021

622

623

221

622

421

021

623

221

622

423

221

422

4

AT00

0420

SSR

201:20

120

3:20

520

120

520

120

320

920

118

920

120

520

120

120

5AU22

3486

SSR

205:20

520

5:21

720

520

820

820

521

720

520

820

520

820

520

820

8AU22

3548

SSR

270:27

827

0:27

026

227

826

227

027

427

027

826

227

026

227

026

227

8AU22

3657

SSR

225:22

522

1:22

522

523

121

922

523

123

322

323

123

1nd

AU22

3670

SSR

194:20

219

4:19

620

219

419

620

219

419

620

219

420

219

420

219

419

620

2nd

ndAU30

1431

SSR

213:21

621

6:21

621

321

621

321

621

321

621

621

321

621

621

321

6AY18

7627

SSR

300:nu

llnu

ll:nu

ll30

030

030

030

0nu

llnu

llnu

llCN44

4542

SSR

136:14

6110:110

136:15

6110:110

128

11012

413

611012

413

611012

614

611012

414

011013

611013

614

611012

413

6

CN44

4636

SSR

241:24

123

9:24

3nd

nd23

924

1nd

23924

124

123

9CN44

4794

SSR

260:30

623

0:29

827

026

023

025

427

025

627

225

427

025

6CN44

5290

SSR

230:23

023

0:23

623

023

023

023

623

024

224

223

624

2CN44

5599

SSR

154:16

413

115

3:17

613

013

213

014

617

613

113

613

113

215

015

413

013

115

213

113

615

413

013

214

615

413

013

114

6

CN49

1050

SSR

>33

0c:c

>33

0a:c

ndnd

>33

0c

>33

0a

>33

0b

>33

0bc

>33

0bc

Tab

le4(con

tinued)

215

SSRname

Fiesta

Discovery

Florina

Nov

aEasaygro

TN10

-8Durello

diForlì

Prima

Mon

dial

Gala

Fuji

CN49

3139

SSR

144:15

612

413

6:15

012

414

212

614

014

216

212

412

614

016

212

414

214

612

414

212

412

614

214

414

014

416

212

414

015

616

2CN49

6002

SSR

243:25

724

3:25

925

726

1nd

24326

124

325

724

325

724

325

924

325

7CN49

6913

SSR

236:23

623

6:24

223

623

623

623

627

823

624

423

625

223

624

4CN58

1493

SSR

194:19

418

4:19

422

819

421

819

222

019

422

819

422

619

421

819

421

8U78

948 S

SR

188:19

018

0:18

6nd

17818

2nd

18418

818

018

6nd

ndU78

949 S

SRb

215x

17620

922

522

1x17

4y20

922

517

620

521

917

421

917

620

721

917

217

619

0211

221

17421

520

519

020

5

Z38

126 S

SR

216:21

621

421

421

621

421

421

624

021

421

421

424

0Z71

980 S

SR

170

170

172

ndnd

172

nd17

0nd

Z71

981 S

SR

222:22

422

4:23

221

222

422

222

422

422

2nd

21622

222

223

2GD14

714

7:15

114

1:15

314

113

515

515

114

714

115

313

515

314

115

314

115

3HGA8b

160:16

4x13

5:15

1y15

6:16

4x15

1:15

1y

13516

015

616

016

413

315

616

015

115

616

016

415

616

413

515

616

016

415

115

616

016

415

115

616

016

4KA4b

141:13

713

9:14

114

113

714

113

714

114

113

713

714

1NB10

2a18

1:nu

ll18

1:18

3nu

ll18

318

3nu

ll18

1nu

ll18

3NH00

9b14

8:16

214

814

414

814

415

813

814

814

415

214

414

815

816

215

816

214

816

2

NH02

9a91

:91

91:93

9199

9795

9395

nd93

95NH03

3b18

9:18

917

5:18

316

317

716

317

717

518

317

918

318

318

917

718

917

718

7MSS6

279:27

927

7:27

327

927

927

327

327

927

927

9

For

theallelesof

FiestaandDiscovery,the

numbersseparatedby

“:”indicatetheestim

ated

size

oftheallelesof

thesamelocus,whiletheothernu

mbersindicatethesize

ofun

definedloci.

For

multilociSSRs,

xand

yindicate

theallelesassign

edto

locusxandy,respectiv

ely

ndallele

size

notdeterm

ined

a Amplicon

sthat

couldbe

allelesof

thelocusxas

wellas

oflocusy.For

theothersevencultivars,thesize

oftheam

plicon

sareindicated;

sing

leallelescanindicate

homozyg

osity

orthe

presence

ofanu

llallele

bDue

tothecomplex

pattern

ofallelesam

plifiedof

thisSSRinsteadof

pairof

alleleson

lyan

alleleperlocuswas

identifiedandispresentedin

thetable(allelethathasbeen

mappedas

ado

minantmarker)

Tab

le4(con

tinued)

216

of these 86 SSRs, 24 (28%) were developed during thisstudy. Some of the selected markers showed a low level ofpolymorphism in our set of reference cultivars. They were,nevertheless, included lacking more polymorphic alternativesfor these specific genomic regions.

Discussion

The aim of our research was to obtain a set of highlypolymorphic SSR markers that cover the entire applegenome, to enable directed genotyping approaches. Di-rected genotyping is a target-directed, cost- and time-efficient approach for the genome-wide genotyping of newcrosses, cultivars, and breeding lines. By facilitating thismethod of genotyping, assessments of new molecularmarker–trait associations, allele mining and validation ofcandidate genes will also be enhanced. This paper reportsthe generation and mapping of a large new set of apple SSR

markers. These SSRs have allowed the enrichment of thereference map of the apple with 168 new loci. This almostdoubled the number of mapped SSR markers.

Efficiency of SSR development

The newly mapped SSRs have been obtained fromdifferent sources: genomic libraries, publicly availableEST sequences, the literature, and from SSRs developedfor other Maloideae species. The efficiency by which eachof these sources gave new SSR markers is evaluated inTable 5. Only about 20% of the library sequences wereunique (not redundant), contained a microsatellite repeat,and were suitable for designing compatible primers in theregions flanking the repeat. When exploiting publishedEST sequences, there is no need to generate enrichedlibraries, or to sequence, and considerable amounts ofmoney and time can be saved.

Table 5 Statistics on SSR development

SSR library/originof the sequence

Sequencedclonesa

Sequences usedfor primer designa

Markersmappedb

Average no. of alleles perorigin of the SSRs

Average no. of repeats perorigin of the SSRsc

GA/GT libraries 571 103 65 (63%) 6.6 28AAGd/AAC/ATCd

libraries587 131 52 (40%) 4.0 9

GenBank 2nt 39 24 (61%) 5.8 14≥3nt 21 7 (33%) 4.3 6Pear and S. torminalise 17 7 (41%) 4.7 naApple SSRs fromliteraturef

8 1 (13%) 13g na

TOTAL 1,158 319 157 (49%)

na: not analyzable; sequences not availableaThe difference between the number of sequenced clones and sequences used for primer design is due to redundant sequences, absenceof a SSR repeat, or a too-short-sequence stretch before or after the SSR repeat for primer designbIn Fiesta × Discovery, or, alternatively in Durello di Forlì × Fiesta, Discovery × TN10-8, or Fuji × Mondial GalacFound in the sequence used for primer designing. Compound SSRs with repeats of different length (e.g., GT-CAA) were notconsidered. Compound SSR with different repeats but with the same number of nucleotides composing the repeat (e.g., GT-TA) wereconsidered and the length of the two repeats was summeddPositive clones of these two libraries were screened by PCR for the presence of highly redundant fragments. The number ofclones sequenced after this check are reportedeYamamoto et al. (2002a,b); Oddou-Muratorio et al. (2001)fHokanson et al. (1998)gThe number of alleles may be overestimated due to low quality of the amplifications

Table 6 Frequency distribution (numbers, percentages, and percentage cumulative) of the number of alleles assessed in a set of nine diploidcultivars of single-locus (SL) SSRs divided by the length of the SSR repeat (two nucleotides or more than two nucleotide repeats)

Absolute no. of SL SSRs No. of alleles

2 3 4 5 6 7 8 9 10 Total

No. of nt repeats 2 6 11 8 10 13 6 9 0 2 65≥3 13 9 13 7 1 3 46

%No. of nt repeats 2 9 17 12 15 20 9 14 0 3 100

≥3 28 20 28 15 2 7 0 0 0 100Cumulative %No. of nt repeats 2 9 26 38 54 74 83 96 96 100

≥3 28 48 76 91 93 100 100 100 100

217

Fig. 1 Genetic map of Fiesta (F) and Discovery (D). Numbering of thelinkage groups is according toMaliepaard et al. (1998), and consistent with

Liebhard et al. 2002, 2003b. SSRs are indicated in bold. SSRs of the newset have been additionally underlined and their map position is also in bold

218

Fig. 1 (continued)

219

Considering the relative high percentage of ESTscontaining SSR repeats (about 5%, data not shown), theefficiency by which SSR containing EST sequences lead to

SSR markers and the continuous increase of publiclyavailable EST sequences (Korban et al. 2005; Crowhurst etal. 2005), it is clear that the next SSR makers should be

220

developed from ESTs. Sequences containing long dinucle-otide repeats are preferable because of their higher level ofpolymorphism (Table 5), making them more valuable indirected genotyping approaches. Other new SSRs may belocated from the mapping of candidate genes that haveSSRs in their non-coding region (e.g., Gao et al. 2005a,b;Costa et al., in preparation), or by the sequencing ofpreviously mapped markers (Gao and Van de Weg 2006).

The approach of transferring SSRs developed in otherMaloideae species was efficient (41%). The high synthenybetween apple and pear was used (Yamamoto et al. 2004;Dondini et al. 2004) to select SSRs, which, from the mapposition in pear, could fill the gaps between SSRs mappedin apple or could enrich regions with few SSRs. Thisstrategy proved to be successful. One of the few new SSRmarkers mapped on LG1 is from pear (KA4b) and someregions (e.g., LG2, LG9, LG16) of the apple genome,which, at the beginning of the project did not contain a highdensity of SSRs, were enriched.

The efficiency of mapping previously developed appleGD-SSRs is probably underestimated due to our stringentPCR conditions (see later). Indeed, Hemmat et al. (2003)were able to map all 18 published GD-SSR markers exceptGD15, which showed very low polymorphism (Hokansonet al. 1998). Ten of these markers could be aligned to ourmap (data not shown), while seven (those tested in thiswork) could not be unequivocally aligned due to a lownumber of markers in common.

Level of polymorphism

The screening of a newly developed SSR over a restrictednumber of cultivars (nine in our case) allows an estimationof its true level of polymorphism. Although the number ofalleles identified is limited compared to what is present inthe apple germplasm, more extensive tests support a roughcorrelation between the allele numbers identified in a smallset of cultivars and the number in germplasm collections(Coart et al. 2003). This information is, thus, useful for afirst selection of markers for genotyping projects as well asfor genetic diversity studies.

New SSRs designed to meet high-throughput criteria

During the development of new SSRs, variable annealingtemperature (Tm) could be used to improve the amplifi-cation profile of individual markers. As our aim was todevelop SSR markers suitable for high-throughput testing,we, therefore, decided to design all primers with a singleTm (60°C), which should enable multiplex PCRs. Therelatively high Tm used was set to improve specificity,avoiding amplification of additional, non-SSR ‘bands’ thatcould hamper both scoring and multiplexing. On the otherhand, some SSR-containing sequences may not havedelivered an SSR marker, although primers for lower Tmmight have been feasible. Tm other than 60°C wereaccepted only for SSR markers developed and mapped inother Maloideae and that had a good chance of filling gapsin the SSR linkage map.

Differences between our standard PCR conditions andprofiles may be the reason why most GD–SSRs couldnot be mapped by us because of very complex bandpatterns that rendered impossible the recognition of theSSR alleles, despite giving good results in other studies(Hokanson et al. 1998, 2001; Hemmat et al. 2003; Vande Weg, unpublished).

Determination of allele sizes

Problems occurring in assessing the size of the amplifiedamplicons of a cultivar, using 33P-labeled primers andpolyacrylamide sequencing gel electrophoresis, have beenalready discussed by Liebhard et al. (2002). The authorsstated that 1) the absolute fragment size could bedetermined only with an accuracy of ±1 base, 2) differencesin size estimation between replications may occur, butrelative size differences among amplicons of testedcultivars are constant. These statements were confirmedin this study. Marker assessments with other technologyplatforms (fluorescently labeled primers, automated se-quencers) will also lead to different absolute values, thoughsize differences should remain constant (This et al. 2004).To allow comparisons among studies, we propose toinclude two or three universal reference cultivars, for whichwe propose Fiesta, Discovery, and Prima. We find thesecultivars suitable because they have been tested with mostapple SSR markers and have been involved in manygenetic studies, being parental cultivars of various mappingpopulations in Europe.

Accuracy of map positions

Although the mapping of the new SSRs is based on theanalysis of 44 progeny plants, their map position can beconsidered to be sufficiently accurate for our purposes. Theorder of the SSRs was usually identical for both theparental Fiesta and Discovery, thus confirming the validityof their relative position (Fig. 1). In only few cases is the

3Fig. 2 Set of 102 SSR primer pairs for global coverage of the applegenome. Map positions (in cM) are aligned to the Discovery mapsof Fig. 1. Gray filled bar segments indicate the linkage groupsegments covered by the Fiesta and Discovery maps of Fig. 1. Openbar segments indicate linkage group segments not covered by themaps of Fig. 1, but which were revealed by other, unpublishedlinkage maps. CH markers mapped by visual alignment that wereinitially mapped in other mapping populations are: CH04c06z[mapped in Prima × Fiesta and Jonathan × Prima (J×P)] andCH02g01 (Discovery × TN10-8 and J×P). For 16 loci, indicatedwith the symbol ?, no primer pairs are publicly available yet. Thesymbol ?* marks positions of unpublished SSR markers, which areexpected to become available in the near future. Underlined SSRshave been developed in the present work

221

order of flanking SSRs inverted. In all these cases, theSSRs involved maps very close together. Relative positionsof tightly linked markers are usually uncertain due to theeffects of missing values and to differences in segregationinformation among markers (Maliepaard et al. 1997). Thedata do allow assessment of approximate map positions ofthe new SSRs, which is sufficient for our current purpose tofill in the gaps in previous maps. In only one case,Z71981SSR (LG15), is the approximate map position still tobe determined. Some SSRs of the Hi set have already beenmapped in other crosses than Fiesta × Discovery, and theirmap position relative to other SSRs was confirmed(Patocchi et al. 2005; Gardiner et al. 2006; Erdin et al.2006; Durel, unpublished).

Genome covering set of apple SSRs

Currently, around 300 SSRs are mapped on the applegenome, mostly in Fiesta × Discovery (Fig. 1). Soon therewill be sufficient SSR markers to enable an initial genome-wide genotyping with a set of 100 SSR markers that havean average inter-marker distance of 15 cM (Fig. 2). Ourgoal is to develop such a set applying the following criteria:(1) distance between successive markers generally notlarger than 20 cM, though occasionally allowed to be up to25 cM; (2) most proximal and most distal marker of alinkage group preferably within 10 cM from the linkagegroup ends; (3) cleanness of the amplifications to alloweasy scoring and multiplexing; (4) high level of polymor-phism; (5) (un)suitability for multiplexing; (6) range of theallele sizes in view of multiplexing markers from the sameLG; (7) preference of CH- over Hi-SSR markers because ofthe generally wider experience with the former; and (8)preference of single-locus markers over multi-locusmarkers for ease of data interpretation.

Applying these criteria, we came up with a set of 86primer pairs, 24 of which (28%) were developed in thecurrent research. This set still lacks markers for 16chromosome segments (Fig. 2); however, in four segments,new SSRs have already been identified in other projects(unpublished data). To fill the remaining gaps, RAPD orAFLP markers previously mapped in these regions couldbe transformed into SCARmarkers, which could be used asprobes to screen apple BAC libraries. From the positiveBACs, sequences containing SSR repeats can be obtainedand used for the development of SSR markers. PCR-basedmethods for the “extraction” of sequences containing SSRrepeats from BAC clones are available (Vinatzer et al.2004).

The current set of 86 SSR markers covers around 85% ofthe genome. This set will be used to genotype 350 cultivarsand breeding selections as well as 1,400 descendants of 24crosses within the framework of the HiDRAS project(Gianfranceschi and Soglio 2004). This work will supplyadditional information on the level of polymorphism ofthese markers and their compatibility in multiplexed PCRs.

SSR database online

Information on the currently available apple SSR markersis scattered over various publications, and publishedinformation remains limited to the initial experienceswith these markers. In the course of their use internation-ally, new information concerning SSR markers will arisewith regard to level of polymorphism, range of allele sizes,number of loci, suitability for multiplexing, etc. World-wide, various groups are also developing new SSR markersfrom the continuously increasing amount of EST data. Forefficient genotyping of cultivars and breeding lines, and foran efficient generation of maps from new crosses, markersshould be sorted according to their map position, poly-morphism, and/or quality. To facilitate searches for andupdates of SSR information, and to concentrate worldwideefforts in the development of new SSR markers, an on-lineapple SSR database was constructed (http://www.hidras.unimi.it). This includes information on all SSR markers ofthe CH (Liebhard et al. 2002), Hi (this paper), and NZ(Guilford et al. 1997; Liebhard et al. 2002) series, and, onceavailable, will include information about the SSRs of theGD series (Hokanson et al. 1998; Hemmat et al. 2003).

This database also allows advanced searches, e.g., a listof SSRs mapped on a specific linkage group, among whichSSRs of a certain quality can be selected. For SSR markerswith amplification profiles of insufficient quality, newprimers can be designed and tested as the sequences of theclones from which the SSR markers were derived can bedownloaded for all CH and Hi markers. Updates andcomments can be added to each of the SSRs. Researchersare encouraged to share their experiences, especially in thereporting of improvements on problematic markers.

Although more than 300 apple SSRs have been mapped,there are regions of the genome not sufficiently coveredwith SSRs (Fig. 2). Saturation of these regions is requiredto allow the construction of maps based solely on SSRs. Inaddition to the method previously proposed, this could beachieved by the further mapping of EST sequencescontaining SSR repeats, as hundreds of such sequencesare available. Such a work could be performed mostefficiently by coordinating action among groups workingwith apple genetic maps all over the world. The currentdatabase is the first step towards the creation of such aworldwide platform. A form is available for announcingthat certain EST sequences are under investigation, so thatduplication of work could be avoided. Contact addressesare also available so that different research groups can getin touch and, if desired, exchange information.

Acknowledgements The authors thank Davide Gobbin andVicente Martinez for the technical assistance. This project is carriedout with the financial support from the Commission of the EuropeanCommunities (Contract No. QLK5-CT-2002-01492), Directorate-General Research—Quality of Life and Management of LivingResources Program. This manuscript does not necessarily reflect theCommission’s views and in no way anticipates its future policy inthis area. Its content is the sole responsibility of the publishers. TheSwiss partner has been financed by BBW No. 020053.

222

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