Development and bin mapping of strawberry genic-SSRs in diploid Fragaria and their transferability...

Development and bin mapping of strawberry genic-SSRsin diploid Fragaria and their transferability acrossthe Rosoideae subfamily

Yasmın Zorrilla-Fontanesi • Amalia Cabeza • Ana M. Torres •

Miguel A. Botella • Victoriano Valpuesta • Amparo Monfort •

Jose F. Sanchez-Sevilla • Iraida Amaya

Received: 4 October 2009 / Accepted: 17 February 2010 / Published online: 20 March 2010

� Springer Science+Business Media B.V. 2010

Abstract Cultivated strawberry (Fragaria 9 ananassa)

together with other economically important genera

such as Rosa (roses) and Rubus (raspberry and

blackberry) belongs to the subfamily Rosoideae.

There is increasing interest in the development of

transferable markers to allow genome comparisons

within the Rosaceae family. In this report, 122 new

genic microsatellite (SSR) markers have been devel-

oped from cultivated strawberry and its diploid

ancestor Fragaria vesca. More than 77% of the

sequences from which the markers were developed

show significant homology to known or predicted

proteins and more than 92% were polymorphic

among strawberry cultivars, representing valuable

markers in transcribed regions of the genome. Sixty-

three SSRs were polymorphic in the diploid Fragaria

reference population and were bin-mapped together

with another five previously reported but unmapped

markers. In total, 72 loci were distributed across the

seven linkage groups. In addition, the transferability

of 174 Fragaria SSRs to the related Rosa and Rubus

genera was investigated, ranging from 28.7% for

genic-SSRs in rose to 16.1% for genomic-SSRs in

raspberry. Among these markers, 33 and 16 were

both localized in the diploid Fragaria reference map

and cross-amplified in rose and raspberry, respec-

tively. These results indicate that transferability of

SSRs across the Rosoideae subfamily is limited.

However, we have identified a set of Fragaria

markers, polymorphic in the diploid reference pop-

ulation, which cross-amplified in both Rosa and

Rubus, which represents a valuable tool for compar-

ative mapping and genetic diversity analyses within

the Rosoideae subfamily.

Keywords Comparative mapping �Synteny � Strawberry � Rose � Raspberry �Rosoideae

Electronic supplementary material The online version ofthis article (doi:10.1007/s11032-010-9417-1) containssupplementary material, which is available to authorized users.

Y. Zorrilla-Fontanesi � A. Cabeza � J. F. Sanchez-

Sevilla � I. Amaya (&)

IFAPA-Centro de Churriana, Cortijo de la Cruz s/n,

Churriana, 29140 Malaga, Spain

e-mail: [email protected]

A. M. Torres

IFAPA-Centro Alameda del Obispo,

14080 Cordoba, Spain

M. A. Botella � V. Valpuesta

Departamento de Biologıa Molecular y Bioquımica,

Universidad de Malaga, 29071 Malaga, Spain

A. Monfort

IRTA-Centre de Recerca en Agrigenomica CSIC-IRTA-

UAB, Carretera de Cabrils Km 2, 08348 Cabrils

(Barcelona), Spain

123

Mol Breeding (2011) 27:137–156

DOI 10.1007/s11032-010-9417-1

http://dx.doi.org/10.1007/s11032-010-9417-1

Introduction

The genus Fragaria belongs to the Rosaceae family

and comprises 23 species, including the commer-

cially important strawberry (Fragaria 9 ananassa)

(Rousseau-Gueutin et al. 2009). The genus displays a

series of ploidy levels, ranging from diploid species

such as Fragaria vesca (2n = 2x = 14) to decaploid

species such as some accessions of Fragaria iturup-

ensis (2n = 10x = 70). The cultivated strawberry,

F. 9 ananassa, is an octoploid (2n = 8x = 56) and

has been the focus of an increasing number of

molecular and genetic studies in recent years (Davis

et al. 2007; Folta and Davis 2006; Hokanson and

Maas 2001). Molecular markers in strawberry have

been developed and used to characterize germplasm

collections (Davis et al. 2006; Gil-Ariza et al. 2009;

Govan et al. 2008) and for genetic mapping (Cekic

et al. 2001; Deng and Davis 2001; Haymes et al.

1997; Lerceteau-Kohler et al. 2004). However, most

of the genetic studies have been performed in

F. vesca, mainly due to its small genome, comparable

to that of Arabidopsis thaliana (Akiyama et al. 2001).

Furthermore, recent data indicate that an ancestor of

F. vesca was the maternal donor of the octoploid

strawberry genome, suggesting a model for the

octoploid genome constitution of Y1Y1Y10Y10ZZZZ,

in which the Y1 and Z genomes would have been

donated from F. vesca and F. iinumae ancestors,

respectively, (Rousseau-Gueutin et al. 2009; Shulaev

et al. 2008). In addition, growing evidence points to a

highly disomic behavior in the commercial octoploid

strawberry, implying a less complex genome than

previously anticipated (Rousseau-Gueutin et al. 2008;

Sargent et al. 2009a).

In species with complex genomes, simple sequence

repeats (SSRs) are the markers of choice in genetic

and breeding studies due to their high polymorphism

and reproducibility, multi-allelic nature and codom-

inant inheritance. They have also proven to be very

useful both for integrating mapping data and compar-

ing maps within and across closely related genera

(Gisbert et al. 2009; Pierantoni et al. 2004; Silfver-

berg-Dilworth et al. 2006). Several studies have

shown a high level of transferability within the

Fragaria genus (Bassil et al. 2006a, b; Davis et al.

2006; Gil-Ariza et al. 2006; Monfort et al. 2006).

Genic-SSRs have been defined as SSRs derived

from genes and expressed sequence tags (ESTs)

representing functional markers in transcribed genes.

This is likely the reason for their higher transferability

across related genera (Arnold et al. 2002; Kuleung

et al. 2004). These and other studies suggested that

genic-SSRs are good candidates for the development

of conserved orthologous markers for genetic analysis

across related genera (Varshney et al. 2005).

The first genetic map of F. 9 ananassa was

developed using amplified fragment length polymor-

phism (AFLP) markers (Lerceteau-Kohler et al.

2003). Although this map has recently been updated

using SSRs, additional markers are needed in order to

saturate the genome of this important crop (Rous-

seau-Gueutin et al. 2008). Very recently, another

genetic linkage map has been developed for

F. 9 ananassa predominantly using SSRs already

mapped in diploid Fragaria (Sargent et al. 2009a).

The first F. vesca map was developed using isoen-

zymes and randomly amplified polymorphic DNA

(RAPD) markers (Davis and Yu 1997). Later, a

transferable linkage map was developed from an

interspecific cross between F. nubicola (recently

reclassified as F. bucharica; Rousseau-Gueutin

et al. 2008) and F. vesca (Sargent et al. 2004). This

map has emerged as the reference map for this genus

and currently contains approximately 340 SSR and

gene-specific markers (Sargent et al. 2008, 2006,

2007; Vilanova et al. 2008).

Bin mapping has proven to be a robust and

efficient approach for genetic mapping (Fernandez-

Silva et al. 2008; Howad et al. 2005; Vision et al.

2000). This strategy improves the efficiency in

saturating genetic maps with molecular markers by

reducing the size of the population. Thus, new

markers are added to an existing linkage map at

lower resolution by genotyping only a subset of

selected, highly informative genotypes (the bin set).

This strategy has recently been developed for diploid

F. vesca and a bin set of only eight plants are

genotyped for an approximate mapping (Sargent et al.

2008), allowing markers or candidate genes to be

time- and cost-effectively located in approximate

positions on the diploid Fragaria reference map. In

this way, a number of evenly distributed markers, or

those located at positions of interest, can be selected

and localized with higher accuracy using large

populations of the octoploid strawberry.

The Rosaceae family, comprising over 90 genera

and 3,000 species, has been recently reorganized into

138 Mol Breeding (2011) 27:137–156

123

three subfamilies: Dryadoideae, Spiraeoideae and

Rosoideae (Potter et al. 2007). The majority of

economically important crops belong to the two latter

subfamilies, with genera such as Prunus, Malus and

Pyrus in the Spiraeoideae and genera Fragaria, Rosa

and Rubus in the Rosoideae subfamily. Comparative

mapping within many plant families has been well

studied: for example, in Brassicaceae, Leguminosae,

Poaceae or Solanaceae (Devos and Gale 2000;

Doganlar et al. 2002; Kalo et al. 2004; Lukens

et al. 2003). These studies allowed the identification

of a marker framework for map-based prediction of

the location of candidate genes involved in agricul-

turally important traits within different species of the

same family. Recently, efforts have been undertaken

for genome comparisons within the Rosaceae (Shu-

laev et al. 2008). A number of reports have analyzed

the transferability of SSRs and other markers across

Rosaceae genera (Decroocq et al. 2003; Gasic et al.

2009; Gisbert et al. 2009; Sargent et al. 2009b, 2007;

Yamamoto et al. 2004). The development and

mapping of transferable markers has allowed the

implementation of synteny studies within the family.

Comparisons between the closely related genomes of

Malus and Pyrus have shown a high level of synteny,

with no major chromosomal rearrangements (Pieran-

toni et al. 2004; Yamamoto et al. 2004). Partial

genome comparison between Malus and Prunus,

which are further apart phylogenetically, has revealed

regions with a high degree of synteny as well as

large-scale chromosomal rearrangements (Dirlewan-

ger et al. 2004). The genomes of the distantly related

Prunus and Fragaria genera have recently been

compared using 71 common markers, revealing low

colinearity between these species (Vilanova et al.

2008). However, to our knowledge, no published

work has yet compared the genomes of strawberry,

rose and/or raspberry, all members of the subfamily

Rosoideae and all of them with a basic chromosomal

number of 7 (2n = 2x = 14).

Initial comparative genetic mapping between

octoploid and diploid Fragaria species has revealed

high levels of macrosynteny and colinearity between

their genomes (Rousseau-Gueutin et al. 2008). Dip-

loid Fragaria maps thus represent a reference for the

genus and might provide a reduced framework of

markers that could be localized in other species such

as the octoploid strawberry. However, due to the

octoploid nature of cultivated strawberry, the number

of transferable markers available for this species is

still limited for comprehensive comparative genetic

analyses. The analysis of transferability of markers

across genera in the Rosoideae subfamily would

identify a set of anchor markers for comparative

mapping. In order to increase the number of available

strawberry SSRs, we have developed 122 new

Fragaria SSR markers, most of them from a

F. 9 ananassa fruit EST collection (Bombarely

et al. submitted) but some of them also from publicly

available F. vesca sequences. All these SSRs were

evaluated for polymorphism between the parental

lines of the reference diploid Fragaria mapping

population and localized in the map. In order to

identify common Rosoideae markers, we analyzed

the transferability of these new SSRs together with 52

other Fragaria SSRs to the closely related Rose and

Rubus genera. Overall, the transferability of SSR

markers was limited within the Rosoideae subfamily,

although a significantly higher cross-amplification

was found among genic-SSRs than among SSRs

developed arbitrarily from SSR-enriched genomic

DNA libraries.

Materials and methods

Plant material and DNA extraction

A total of 14 strawberry cultivars was used to

estimate the polymorphism of the newly developed

SSR markers in F. 9 ananassa (Table 1). These

included Chandler and Carisma as source of the

majority of EST-SSRs, IFAPA selections 232 and

1,392, and cultivars Parker, Endurance and Selva as

parental lines of different strawberry mapping pop-

ulations and cultivars Festival, Cuesta, Solut, Josif

Mahomed, Camarosa, Fern and Toyonoka covering

further genetic variation. For each primer pair at least

six different accessions were used in the polymor-

phism evaluation.

To evaluate the amplification and polymorphism

in diploid Fragaria, up to 8 accessions were analyzed

(Table 1). Genomic DNA from all Fragaria acces-

sions was extracted from young leaf tissue using the

DNeasy plant miniprep kit (Qiagen) according to the

manufacturer’s protocol.

To analyze the transferability and polymorphism

in rose, we evaluated two species, the hybrid Rosa

Mol Breeding (2011) 27:137–156 139

123

moschata 9 Rosa chinensis Blush Noisette (D10)

and Rosa wichurana (E15). These species are the

parental lines of the F1 mapping population analyzed

by Dugo et al. (2005; Table 1). DNA was isolated

according to Torres et al. (1993), replacing the

extraction buffer by one adapted to woody species

(Cheng et al. 1997).

To evaluate the transferability of Fragaria SSRs to

raspberry (Rubus idaeus), young leaves were col-

lected from cultivars Tulameen and Miraz growing in

pots at the IFAPA-Centro de Churriana germplasm

collection (Table 1). DNA was extracted using a

modified CTAB method based on that of Doyle and

Doyle (1990).

SSR selection and analysis

The new Fragaria SSR markers developed in this

study were obtained from two different sources. The

F. 9 ananassa EST-SSRs were designed from the

FREST strawberry database, comprising ESTs from

cDNA libraries of different developmental stages of

strawberry fruit (Bombareli et al. submitted).

Approximately 10,000 of these ESTs were grouped

as either contigs or singletons and analyzed for the

presence of SSR motifs using the PERL5 script

MISA (Thiel et al. 2003). The F. vesca SSRs were

obtained from public sequences in the EMBL data-

base. A total of 150 flanking primers were designed

Table 1 Plant material

used for marker

polymorphism, mapping

and cross-species

transferability

a Source of accession

numbers: FDP, East

Malling research, UK;

IFAPA, IFAPA-Centro de

Churriana, Spain; PI,

National clonal germplasm

repository corvallis, USA;

D10 and E15, IFAPA-

Alameda del Obispo

Species Primary denomination/accession no.a Origin

Fragaria

F. 9 ananassa Chandler USA

Carisma Spain

232 Spain

1392 Spain

Parker USA

Endurance USA

Selva USA

Festival USA

Cuesta USA

Salut Poland

Josif Mahomed Russia

Camarosa USA

Fern USA

Tonoyoka Japan

F. vesca F. vesca spp. vesca f. semperflorens/FDP 815 N.A.

IFAPA 14 Spain

F. vesca ssp. bracteata/IFAPA 184/= PI 551514 USA

Reine des vallees/IFAPA 660 France

F. vesca spp. vesca f. alba/IFAPA 596 Italy

F. bucharica FDP 601 Pakistan

F. iinumae PI 637963 Japan

PI 637966 Japan

Rosa

R. moschata 9 R. chinensis Blush Noisette/D10 USA

R. wichurana Basye’s Thornless/E15 China

Rubus

R. idaeus Tulameen Canada

Miraz Russia

140 Mol Breeding (2011) 27:137–156

123

using the PRIMER3 program (Rozen and Skaletsky

2000). Primers were designed to have an annealing

temperature (Ta) of 60–62�C, a length of 20–22 bases

and generate product sizes ranging from 100 to

250 bp in length. Functional annotation of ESTs was

performed using BLAST and only homologies below

an E-value of 1e-10 were considered significant.

SSR amplifications were performed in a BioRad

iCycler in a final volume of 15 ll, containing

1 9 PCR buffer (GeneCraft; 16 mM (NH4)2SO4,

67 mM Tris–HCl pH 8.8, 0.1% Tween 20), 2 mM

MgCl2, 200 lM each dNTPs, 0.2 lM of each specific

primer, 0.5 U of Taq DNA polymerase (GeneCraft)

and 25 ng of genomic DNA. The adopted PCR profile

was as follows: 94�C for 3 min, then 35 cycles of

94�C for 30 s, annealing at 57–65�C for 30 s and

extension at 72�C for 45 s. A final extension was

carried out at 72�C for 3 min.

Initially, SSR validation and annealing temperature

was analyzed using template DNA from F. 9 anan-

assa cv. Chandler or F. vesca 815 and a gradient PCR

with Ta ranging from 55 to 65�C. PCR products were

separated in 1.5% agarose gels and stained with

ethidium bromide. Those primer pairs showing clear

products of approximately the expected size were

selected and used for further analyses. When primer

pairs failed to amplify or the amplification product was

weak, a modified Taguchi method was applied in order

to optimise the conditions (Cobb and Clarkson 1994).

The reaction components evaluated in the orthogonal

array were the amount of DNA template, primers,

dNTPs and MgCl2. Each component was used at three

predetermined levels, each of which occurred an equal

number of times within the orthogonal array. This

resulted in only nine different reaction combinations

that were set up in duplicate. After separation of PCR

products in 1.5% agarose and staining with ethidium

bromide, an image was obtained using a Gel Doc EQ

system (BioRad) and the relative band intensities were

estimated using the Quantity one software (BioRad).

Average band intensities for each of the nine reactions

were subsequently used to determine the different

component effects and to estimate the optimum

reaction conditions according to the method of Cobb

and Clarkson (1994). Optimized PCR conditions for

primer pairs with initial weak product amplification

are shown in supplementary Table 1S.

For polymorphism evaluation and mapping, PCR

products were mixed with 10 lL of formamide

loading buffer (98% formamide, 10 mM EDTA and

0.1% xylene cyanol), denatured at 95�C for 3 min

and immediately cooled on ice. Two to four micro-

litres of each sample were separated by electropho-

resis in 4.5% denaturing polyacrylamide gels at a

constant power of 95 W for 1–3 h. The fragments

were visualized by silver staining according to the

protocol of Benbouza et al. (2006). Allele sizes were

determined by comparison to the 10-bp DNA ladder

(Invitrogen, San Diego, CA, USA).

The number of visible alleles per cultivar and the

total number of alleles were evaluated by visual

inspection in the analyzed cultivars. The effect of

SSR type and length on the number of alleles was

analyzed using the STATISTICA 7.0 software (Stat-

Soft, Inc. 2007). A t-test was used to compare the

mean number of alleles of polymorphic di- and

trinucleotides. The correlation between SSR length

and total number of alleles was studied by linear

regression analysis, both globally and separately for

di- and trinucleotide repeats.

Bin mapping in the diploid Fragaria reference

map (FV 9 FB)

The F. vesca 815 parental line, the F1 hybrid and the

six bin set seedlings were genotyped with the novel

polymorphic markers as described in Sargent et al.

(2008). In addition, we found polymorphisms in five

public SSR markers not previously mapped in the

diploid reference map. These markers were

ChFaM004, ChFaM005 and ChFaM007 from Gil-

Ariza et al. (2006), Fvi9 developed from F. virginiana

(Ashley et al. 2003) and PBCESSRFXA12 reported by

Keniry et al. (2006). Markers were assigned to bins by

visually matching the joint genotype obtained to those

defining the different Fragaria bins. An additional

seedling (31) was genotyped when the marker was

assigned to the redundant bins II:50 and III:53. Gel

bands were scored by two different observers. Markers

that did not correspond to any of the described bins

were subsequently scored in the entire FV 9 FB

population (94 seedlings) to assign a precise position

to both the marker and the novel bin. In this case,

linkage analysis and map construction was performed

as described by Sargent et al. (2006). Dominant

markers, which have a joint genotype matching several

bins, were mapped in additional individuals in order to

be assigned to single bins.

Mol Breeding (2011) 27:137–156 141

123

SSR amplification in Rosa and Rubus

Newly developed genic-SSRs together with SSRs

already developed from Fragaria were used to

amplify DNA from the hybrid Rosa moschat-

a 9 Rosa chinensis Blush Noisette (accession D10)

and Rosa wichurana (accession E15), the parents of

the rose mapping population analyzed by Dugo et al.

(2005) and two accessions of raspberry, Tulameen

and Miraz. Amplification reactions were carried out

identically to those for Fragaria, the only exception

being to decrease to 60�C the Ta of primer pairs when

it was above that value. Amplification and polymor-

phism was evaluated in polyacrylamide gels and

silver staining as described above.

Results

Development of novel Fragaria SSR markers

Recently, the number of available F. 9 ananassa

DNA sequences has increased and around 10,000

genes and ESTs from strawberry are available in

public databases (Bombarely et al., submitted;

http://www.fresa.uco.uma.es). A search for SSR

motifs within these sequences allowed the identifi-

cation of 383 SSRs in 329 of the 7,096 unigenes

analyzed. Subtracting sequences in which SSR

markers had been already developed and sequences in

which the SSR motifs were too close to the end, 187

sequences could be used for marker development. A

total of 150 primer pairs, originating from either

contigs or singletons, were designed. One hundred

and forty-eight primer pairs were designed from

ESTs of F. 9 ananassa fruit libraries and F. vesca

EST sequences available in the Genbank database

while only two (ChFaM148 and ChFvM125) were

designed from genomic sequences within and adja-

cent to a gene respectively, thus representing markers

of putative functional genes. These primers were

initially tested in the genomic DNA of F. 9 anan-

assa cv. Chandler and F. vesca 815 and 112 primer

pairs rendered strong amplification products. The

remaining 38 primer pairs that either failed to amplify

or produced a faint band were then optimized for

PCR amplification using the Taguchi method (Cobb

and Clarkson 1994), and selected conditions are

specified in supplementary Table 1S. Finally, a total

of 138 primer pairs (92%) rendered clear amplifica-

tion products, with 122 (88.4%) producing amplicons

of sizes approximately equal to those expected in

silico. Since all but two of the sequences used for

primer design were ESTs, the failure of amplification

or the amplification of larger products could be

explained by the presence of introns. Of the 122 new

SSR markers developed, 100 were developed from

cultivated strawberry sequences (Table 2) whereas 22

were developed from F. vesca (Table 3). Among

these genic-SSRs, 36.9% were dinucleotide repeats,

55.7% were trinucleotide repeats, 4.9% contained

both di- and trinucleotide repeats and one and two

were tetra- and pentanucleotide repeats, respectively.

For the EST sequences, homology to genes with

known function could be established for 81 sequences

(67.5%), with 12 markers (10%) in unknown proteins

and 27 sequences (22.5%) with no significant simi-

larity found (Tables 2 and 3).

Polymorphism in F. 9 ananassa was evaluated in

6–10 cultivars selected because of (1) being a source of

ESTs, (2) their diverse origin or (3) being parental

lines of various strawberry mapping populations

developed in our research centre (Table 1). Only 9

out of the 122 SSR markers (7.4%) did not show

polymorphism in the analyzed genotypes. Interest-

ingly, in all cases the SSR motif was a trinucleotide

repeated 5–6 times localized in the open reading frame

(ORF; Table 2). It has to be noted, however, that 23

other SSR markers were polymorphic even though the

SSR motif (trinucleotide repeats in all instances) was

located in the ORF. The majority of the polymorphic

SSR markers generated more than two bands in each

cultivar, representing different alleles at a choice of the

four genomes of octoploid strawberry. However, 16

SSR markers (14.2%) amplified only one or two bands

in each of the analyzed cultivars, suggesting that they

may be specific to one genome. To confirm this, a

larger representation of cultivars should be analyzed

and the inheritance behaviour tested in an octoploid

strawberry segregating population.

Based on the number of amplified alleles, trinu-

cleotide motifs were found to be as equally poly-

morphic as dinucleotide motifs, as the mean number

of alleles generated by polymorphic di- or trinucleo-

tide SSRs (6.29 and 5.27, respectively) was not

significantly different (p = 0.06). As an example, the

four most polymorphic SSR markers generated a total

of 14–15 alleles in the analyzed cultivars, with two of

142 Mol Breeding (2011) 27:137–156

123

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CC

TT

GC

AC

CG

AT

TC

hF

aM01

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U81

5789

(TC

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C)5

N.P

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GC

CG

CA

TC

CC

TC

TT

TT

CT

A62

217

N.S

.+/

+19

8-22

98

6-7

+/-

210

-/-

-+/

+20

7-21

7R

:C

TA

GG

GA

TT

GA

GG

AC

CG

AC

AC

hF

aM02

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5790

(AG

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F:

GG

GC

CA

CT

CC

TA

CT

TC

TT

CA

6121

2L-

Asp

arag

inas

e+/

+19

8-26

03

2-3

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--/

--

-/-

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:T

TG

GC

CT

TG

AG

AG

CT

TC

GA

TC

hF

aM02

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U81

5791

(AA

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UT

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TC

TC

TC

GT

TT

CT

CT

AA

AA

GC

5811

3P

rofil

in+/

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53-

5+/

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R:

AT

CA

AG

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8157

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CT

CT

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176

Thi

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1-2

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AA

AT

CC

CG

AG

AG

CC

TT

TC

hF

aM03

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5793

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A)8

OR

FF

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AA

GC

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TA

AG

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A58

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0A

vr9/

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9el

icite

dpr

otei

n14

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+18

0-18

54

2-4

+/-

182

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--/

--

R:

AC

GG

TG

GG

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CA

CT

TA

AA

GA

Ch

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032

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8157

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GG

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T59

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N.S

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7-22

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2-3

+/-

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--

R:

TT

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GC

CC

CA

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TT

CC

AG

TA

Ch

FaM

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8157

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TT

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Hyd

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2-3

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325-

350

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AG

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9C

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5-21

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177-

190

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230-

255

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TC

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GA

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8157

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196-

204

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CT

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hF

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U81

5799

(CT

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OR

FF

:G

TG

GT

TT

TT

GT

TG

GG

CA

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G60

184

Mito

chon

dria

lcar

rier

prot

ein

+/+

575-

600

32-

3+/

-57

5-/

--

-/-

-R

:A

AC

GG

CT

TA

TC

AT

CC

TG

CA

TC

hF

aM04

0G

U81

5800

(TC

C)7

OR

FF

:A

GT

GG

TC

AT

CA

GC

AC

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A62

194

60S

ribos

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ein

L18a

+/+

180-

210

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4+/

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-/

--

--

R:

TA

AC

CG

GG

AA

CG

GT

AC

TC

TG

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FaM

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8158

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AG

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F:

CC

AC

AC

GA

AG

GA

GA

AG

GA

GA

6018

6R

NA

ornu

clei

cac

idbi

ndin

gpr

otei

n+/

+16

5-19

08

2-6

+/-

185

-/-

--/

--

R:

AA

CG

TG

TC

AT

TC

GA

CC

AC

AA

Ch

FaM

044

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8158

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CC

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GA

GT

CG

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CT

AA

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7-22

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R:

TT

TT

GT

TG

AC

GA

GC

GA

GA

TG

Ch

FaM

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8158

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CT

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F:

CC

AT

TT

CC

AT

GG

CC

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TT

6113

4Li

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otei

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+13

4-15

45

3-5

+/-

100-

135

-/-

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-11

7R

:G

GC

CT

TG

TT

GG

GT

CT

GA

GA

GC

hF

aM04

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U81

5804

(TC

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F:

TC

AT

TT

CT

CT

CT

CC

CC

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GA

T60

207

N.S

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+21

2-22

07

3-4

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--/

--

-/-

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AT

GT

GA

TT

TA

CG

GC

GA

AG

GC

hF

aM05

6G

U81

5805

(GC

A)6

OR

FF

:A

AA

AC

GT

CG

TC

GT

TC

AG

GA

T58

208

Nuc

lear

acid

bind

ing

prot

ein

+/+

202-

211

31-

2+/

-20

5+/

-29

0+/

+19

0-19

5R

:C

GT

AC

TG

CT

GT

TG

CT

GC

TG

TC

hF

aM05

8G

U81

5806

(AC

C)6

OR

FF

:G

AC

CC

AA

AC

AC

AC

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‡16

2G

row

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ctor

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162-

172

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3+/

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0+/

-15

2+/

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5R

:C

TC

CC

CA

TT

CT

GG

GA

AA

TC

TC

hF

aM06

0G

U81

5807

(CC

G)6

N.P

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:T

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GC

TA

CC

AC

CA

AG

AA

CC

C58

‡20

1N

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+/+

197-

209

42-

3+/

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3-/

--

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AT

AC

CC

TT

GG

TA

CC

CC

TC

GC

hF

aM06

1G

U81

5808

(CT

T)6

N.P

.F

:G

TG

CT

CA

AG

AA

AC

CC

TT

TC

G58

203

N.S

.+/

+19

0-22

08

2-5

+/+

190

-/-

-+/

+15

9-16

1R

:G

CG

CT

AG

CA

AC

AG

TA

AG

GT

GC

hF

aM06

2G

U81

5809

(TG

A)5

...(T

TG

)63´

UT

RF

:G

GT

TT

CG

CT

TG

GA

GG

AT

AT

G58

167

Zin

c-fin

ger

prot

ein

+/+

1551

624

3-4

+/+

160-

170

-/-

--/

--

R:

CA

CC

AA

AT

CT

CT

GA

TC

AC

AA

AA

AC

hF

aM06

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U81

5810

(CC

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OR

FF

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AC

GT

CT

CC

GA

TC

CG

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GA

T61

156

Cys

tein

epr

otei

nase

RD

19A

+/-

148-

158

22

+/-

158

+/+

160-

166

-/-

-

Mol Breeding (2011) 27:137–156 143

123

Ta

ble

2co

nti

nu

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F.×

an

anas

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Ro

s aR

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cus

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Ch

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5817

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prot

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163-

182

53-

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AC

TC

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A58

162

Unk

now

npr

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86

3-6

+/+

160-

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R:

AT

AG

AA

GC

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GC

GT

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TG

Ch

FaM

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8158

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CC

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GA

AG

AA

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G58

232

Put

ativ

eE

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4pr

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3-4

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CG

AA

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Ch

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8158

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168-

179

43-

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CT

CT

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U81

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CC

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C58

155

serin

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reon

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prot

ein

kina

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+15

2-16

03

1-3

+/-

154

+/+

220-

232

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CG

AC

CA

TT

CC

TC

TT

CC

TC

AC

hF

aM06

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U81

5816

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OR

FF

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GA

CC

CT

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GC

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T59

‡14

6R

NA

-bin

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prot

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+/-

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11

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--

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CA

GG

CA

AT

TC

CT

AA

CT

Ch

FaM

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8158

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TG

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F:

AG

CA

TT

GA

AT

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CC

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1S

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prot

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+/-

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11

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--

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193-

196

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CC

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GC

AG

CA

GG

AA

CT

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Ch

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A58

143

TIN

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146-

152

31-

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GC

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5722

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Pas

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--

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CC

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8158

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CA

GC

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CA

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+/+

249-

255

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2+/

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TC

TA

CG

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6019

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AU

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06

2-5

+/-

200

+/-

202

+/-

217

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AT

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CA

CT

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Unk

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GC

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oxid

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191

11

+/-

198

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181

11

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194-

198

+/-

195

+/-

188

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Pur

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4U

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53-

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06

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21-

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--

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126

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--

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AT

GG

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AG

CT

TG

GA

CT

TT

CC

hF

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5835

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UT

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CC

AG

AA

GC

AA

AA

AC

CA

GA

A58

164

Sig

nalp

eptid

ase

prot

ein

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138-

170

74-

6+/

+16

8-17

0-/

--

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:G

GG

AA

GT

TG

AA

AT

TG

TC

GG

AC

hF

aM09

7G

U81

5836

(TC

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UT

RF

:G

CC

AA

AG

GT

TG

GT

TC

TT

TG

A58

236

Unk

now

npr

otei

n+/

+23

6-26

06

1-3

+/-

280

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--

R:

GC

CA

TT

TT

GG

AA

GA

GG

TG

AA

Ch

FaM

098

GU

8158

37(T

C)7

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TR

F:

GT

GA

GA

GT

CA

GC

CC

AC

CC

TA

5821

4U

nkno

wn

prot

ein

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190-

270

143-

6+/

+21

8-22

0+/

+35

0-40

0-/

--

144 Mol Breeding (2011) 27:137–156

123

Ta

ble

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nti

nu

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ence

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GC

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T)7

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101

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8158

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G)7

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TA

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T58

157

3-M

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R:

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T)7

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152-

162

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CG

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104

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8158

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AA

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8158

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G)7

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Ser

ine/

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142

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109

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8158

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R:

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5850

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UT

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GT

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TG

CC

TT

GG

TT

TA

CA

A62

209

Mem

bran

ein

trin

sic

prot

ein

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248

31-

3+/

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0-23

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CG

CC

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TC

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CT

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136

Unk

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npr

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0-65

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650

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350-

450

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R:

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AC

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TC

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ike

prot

ein

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202

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186

R:

GA

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AG

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GT

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120*

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8158

53(T

TC

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AG

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175-

185

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180-

190

R:

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AA

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122

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8158

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TC

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min

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lect

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144

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A61

191

Abs

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AT

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6019

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202

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225

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142

Mon

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144

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188

Ter

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210

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177

R:

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-

Mol Breeding (2011) 27:137–156 145

123

Ta

ble

2co

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nu

ed

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anas

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261

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AA

AA

6015

6R

NA

-bin

ding

prot

ein

+/+

118-

158

94-

6+/

+15

0-15

5-/

--

-/-

-R

:A

GC

GC

AT

GA

GT

CT

GC

TG

TT

AC

hF

aM20

3C

O38

0455

(GA

)17

N.P

.F

:C

GA

GG

GT

CA

CG

GC

TA

CT

AA

A58

169

N.S

.+/

+15

0-21

05

3-4

+/-

150

+/+

160-

174

+/+

162-

168

R:

TG

AG

TA

TG

AC

CA

AT

CC

GA

AA

GA

Ch

FaM

208*

GU

8158

71(C

TT

)55´

UT

RF

:T

TT

CT

CT

CT

TC

TC

TC

TC

TC

TC

AC

TG

5622

8S

GT

1re

late

dpr

otei

n+/

+23

2-24

04

1-2

+/+

232-

235

+/+

185-

285

+/-

260

R:

TC

GG

TG

AG

AT

TG

TT

GG

AT

TT

Ch

FaM

209

GU

8158

72(C

TC

TT

)45´

UT

RF

:C

CC

CC

AA

AA

CC

CT

CT

AT

TC

T60

144

Hom

eobo

xtr

ansc

riptio

nfa

ctor

+/+

135-

155

43-

4+/

+12

4-14

2+/

+14

5-16

0-/

--

R:

AT

CA

TT

CC

CA

AG

CC

AC

TG

TC

Ch

FaM

214

GU

8158

73(A

GA

)55´

UT

RF

:C

TC

AC

CA

CA

AC

TG

CA

AC

AC

C59

169

Mem

bran

ech

anne

lpro

tein

+/+

153-

186

62-

4+/

-16

2-/

--

-/-

-R

:T

GA

TA

TT

GA

GC

AA

CG

GG

TG

A

*Tra

nsfe

rabl

em

arke

rsac

ross

the

Ros

oide

aege

nera

map

ped

inF

raga

ria.

‡ Mar

kers

with

optim

ized

PCR

cond

ition

s(T

able

1S).

a Loc

atio

nof

the

SSR

with

resp

ectt

oth

ege

ne.W

hen

itha

sno

tbee

npo

ssib

leto

dete

rmin

e,N

.P.i

sin

dica

ted.

b The

expe

cted

PCR

prod

ucts

ize

inba

sepa

irs

base

don

the

sequ

ence

from

whi

chea

chpr

imer

pair

isde

rive

d.c T

otal

num

ber

ofba

nds

iden

tifie

din

the

tota

lnum

ber

ofan

alyz

edcu

ltiva

rs.

d Num

ber

ofba

nds

iden

tifie

din

each

cult

ivar

.e A

mpl

ific

atio

nan

dpo

lym

orph

ism

inth

epa

rent

allin

esof

the

dipl

oid

Fra

gari

are

fere

nce

segr

egat

ing

popu

latio

n.W

hen

apr

imer

pair

ampl

ifie

da

prod

ucti

nan

yge

noty

pe,a

‘+’

sign

isin

dica

ted.

Ifa

prim

erpa

iral

sode

tect

edpo

lym

orph

ism

sa

‘+/+

’sig

nis

indi

cate

d.W

hen

prim

erpa

irs

did

nota

mpl

ify

inF

.ves

cabu

tam

plif

ied

apr

oduc

tin

F.b

ucha

rica

,a‘-

/+’

sign

isin

dica

ted.

f Am

plif

icat

ion

and

poly

mor

phis

min

Ros

aw

ichu

rana

(E15

)an

d‘B

lush

Noi

sett

e’(D

10).

g Am

plif

icat

ion

and

poly

mor

phis

min

Rub

usid

aeus

cult

ivar

s‘T

ulam

een’

(Rt)

and

‘Mir

az’

(Rm

).

146 Mol Breeding (2011) 27:137–156

123

Ta

ble

3C

har

acte

rist

ics

of

22

F.

vesc

aS

SR

mar

ker

sd

evel

op

edin

this

inv

esti

gat

ion

and

amp

lifi

cati

on

inF

.9

an

an

ass

a,

dip

loid

Fra

ga

ria

refe

ren

cep

op

ula

tio

n,

dip

loid

Ro

saan

dR

ub

us

F.×

an

anas

saF

rag

aria

Ro

saR

ub

us

Lo

cus

Acc

essi

on

Mo

tif

Po

siti

on

aP

rim

erse

qu

ence

(5'-

3')

Ta

(ºC

)S

izeb

Pu

tati

vefu

nct

ion

Cu

ltiv

ars

Siz

era

ng

eN

o.

alle

lesc

Alle

les/

cvd

FV

xF

Be

Siz

era

ng

eE

15/D

10f

Siz

era

ng

eR

t/R

mg

Siz

era

ng

eC

hF

vM02

8D

V43

8195

(CT

T)6

N.P

.F

:AA

TG

GC

AT

CA

AC

TT

CT

GC

AC

5817

5U

nkno

wn

prot

ein

+/+

160-

180

54-

5+

/+17

3-17

6+/

+17

5-18

5-/

R:C

AG

CC

TG

CT

GC

TG

TA

GT

TC

TC

hF

vM04

9E

X67

1238

(AA

G)7

3Ú

TR

F:A

TG

GT

TG

GT

GA

TC

AA

TG

GT

G58

159

Met

alio

nbi

ndin

gpr

otei

n+/

+15

1-17

37

3-4

+/+

156-

160

-/-

--/

R:T

TC

AT

AT

GC

AA

TT

TG

AT

GG

AA

CC

hF

vM05

0D

Y67

4195

(TC

)22

5Ú

TR

F:C

CC

TT

CC

CT

TT

CC

TT

TC

CT

T57

‡15

6F

e-S

met

abol

ism

asso

ciat

edpr

otei

n+/

+12

0-15

611

5-8

+/+

140-

150

-/-

--/

R:T

AA

TG

GG

GT

GG

TG

GA

GT

TC

AC

hF

vM07

5D

Y67

5833

(AA

C)9

N.P

.F

:AG

GA

AG

AG

GA

CG

AG

GA

CG

AA

6122

6R

3Hdo

mai

npr

otei

n+/

+42

0-47

02

1-2

+/+

460-

470

-/-

--/

R:A

CC

TG

CA

TT

CC

CT

CC

TG

AT

TC

hF

vM08

4D

Y67

5569

(TT

GA

)5(G

A)6

5Ú

TR

F:C

CA

AA

CA

GT

CC

TC

AC

GG

TT

C61

151

RIN

G-H

2fin

ger

prot

ein

+/+

150-

158

42-

4+/

-15

4-/

--

-/R

:GT

AG

CA

CC

AG

CA

AC

AT

GA

CG

Ch

FvM

087

DY

6753

66(G

AA

)95´

UT

RF

:GA

AA

GG

GG

AA

GC

CT

TT

TC

AT

6022

4A

cid

phos

phat

ase

+/+

208-

239

63-

5+

/+21

2-23

2-/

--

-/R

:TG

GG

AC

GA

AA

GT

TC

CC

AA

TA

Ch

FvM

125

EU

0248

61(A

G)9

fosm

idF

:GC

GA

CT

GC

CA

TC

GT

AA

CC

TA

6016

710

Kb

ofC

ON

ST

AN

S+/

+16

0-21

04

2-/

+17

5-/

--

-/R

:TC

TC

CA

AT

GT

TT

TC

GC

CA

AG

Ch

FvM

140

DY

6745

56(T

C)1

65´

UT

RF

:CC

AC

TT

CC

CA

TA

CC

AC

TG

GA

6113

1S

ucro

secl

eava

gepr

otei

n+/

+94

-137

122-

10+

/+12

2-13

7+/

+13

7-18

7-/

R:

CG

TC

GT

AG

GT

TC

GG

TC

TC

TG

Ch

FvM

181

DY

6683

43(G

GT

)5cg

c(G

GT

)6N

.P.

F:G

GA

GA

AC

TG

CT

TT

GG

TA

GG

G59

‡15

3N

.S.

+/+

124-

178

73-

5+

/+16

0-17

0-/

--

-/R

:AC

GT

AC

AC

CA

GC

AG

AT

GA

GC

Ch

FvM

182

DY

6733

63(C

AA

)6...

(AC

A)5

5Ú

TR

F:G

GA

AC

CA

AA

CG

AA

CA

CC

AA

C63

210

AT

-Hoo

kD

NA

-Bin

ding

Pro

tein

+/+

190-

240

74-

6+

/+23

0-23

6-/

--

-/R

:GC

GG

AG

GA

GT

GA

GT

GA

AG

AC

Ch

FvM

184

DY

6678

64(G

AT

)12g

gtgg

(TG

A)6

OR

FF

:GC

AC

TT

TG

GG

GT

TT

TG

GG

TA

6124

4F

-box

fam

ilypr

otei

n+/

+19

8-24

89

3-5

+/+

228-

240

-/-

--/

R:T

TG

CA

AG

AA

CC

CC

CT

TC

CT

AC

hF

vM19

1D

Y67

3483

(CA

A)7

(CA

G)1

5O

RF

F:C

CA

GC

AG

AA

TC

CT

CA

AA

TG

C61

231

WD

-40

repe

atpr

otei

n(S

TY

LOS

A)

+/+

205-

255

83-

4+

/+20

0-24

0+/

+21

0-23

5-/

R:G

TG

CC

AA

CA

AG

CC

CA

TT

GC

hF

vM19

2D

Y66

8080

(AC

A)5

...(C

CA

)5O

RF

F:T

GA

GG

TC

AG

TC

TG

TG

AG

AA

TT

G57

‡20

0bH

LHtr

ansc

riptio

nfa

ctor

+/+

198-

285

146-

10+/

-20

8-/

--

+/+

700-

800

R:T

TT

CC

GA

GA

GT

GG

AG

AG

CA

TC

hF

vM19

3D

Y66

9902

(AT

C)5

(TC

)21

N.P

.F

:CA

TC

AG

AA

CC

AT

CA

AT

CA

TC

G56

‡17

8N

.S.

+/+

166-

182

63-

6+

/+17

5-18

5+/

+18

5-21

8-/

R:T

AC

TG

CC

GG

AG

AA

TG

AA

CA

Ch

FvM

201

CX

6619

12(T

C)1

05´

UT

RF

:TG

AT

TC

AA

CT

CC

AG

CG

AA

GC

6116

1A

nkyr

indo

mai

npr

otei

n+/

+22

0-28

09

4-7

+/+

260-

270

-/-

--/

R:A

TC

AG

CA

GG

CG

AA

TC

CT

TC

TC

hF

vM20

2D

Y67

3477

(GA

)21

5Ú

TR

F:C

AA

AG

GG

CT

CC

AG

CT

AT

CT

C62

185

Fat

tyac

idhy

drox

ylas

e+/

+15

0-19

06

3-5

+/+

195-

200

-/-

--/

R:A

GG

AT

CG

TC

AG

CT

AA

GA

AG

CA

Ch

FvM

205

DY

6720

89(A

TT

)14

3Ú

TR

F:G

CG

AA

AC

CC

TA

TG

GA

TT

GT

T61

152

Pep

tidyl

-pro

lylc

is/tr

ans

isom

eras

e+/

+11

2-17

26

3-4

+/+

130-

153

-/-

--/

R:A

CA

AC

CA

CC

AA

AT

TC

CC

AT

TC

hF

vM20

6D

Y67

5467

(CT

)19

5Ú

TR

F:C

CG

AG

AG

AG

CC

AT

AA

CC

AC

T57

‡15

4N

ucle

icac

idbi

ndin

gpr

otei

n+/

+20

0-23

06

2-4

+/+

235-

250

+/+

218-

238

-/R

:GT

TT

AT

AG

GC

GT

GG

GC

TC

TG

Ch

FvM

207

DY

6717

69(A

GA

)12

5Ú

TR

F:T

AG

TC

GC

CC

CA

TT

CC

TT

TC

T61

133

Ank

yrin

repe

atfa

mily

prot

ein

+/+

500-

650

54-

5+/

-55

0-/

--

-/R

:AC

AG

AG

CA

CC

AA

AC

CC

AC

AC

Ch

FvM

210

DY

6750

18(T

C)2

35´

UT

RF

:TC

CC

CA

CA

TT

TT

CC

TT

GT

TT

5922

0A

uxin

-reg

ula

ted

prot

ein

+/+

190-

220

52-

5+

/+18

0-19

2-/

--

-/R

:GT

GG

TG

TT

GG

TG

AG

TG

GA

GA

Ch

FvM

212

DY

6737

12(T

CT

)75´

UT

RF

:CA

AA

TC

TT

CA

AC

GG

TC

TC

TC

C60

115

Tra

nske

tola

se+/

+95

-135

63-

6+

/+11

8-14

0-/

--

-/

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

R:A

CG

GA

GG

AG

GA

GG

AA

GT

CA

TC

hF

vM21

3*D

Y67

2560

(GA

)95´

UT

RF

:AA

CC

CT

AG

GA

GG

CT

GA

AA

CC

6021

560

SR

ibos

omal

prot

ein

L27A

+/+

218-

250

42-

3+

/+22

0-23

6+/

-21

5+/

-21

5R

:AT

AC

CC

CG

GG

TG

GT

AC

TT

GT

*Tra

nsfe

rabl

em

arke

rsac

ross

the

Ros

oide

aege

nera

map

ped

inF

raga

ria.

‡ Mar

kers

with

optim

ized

PCR

cond

ition

s(T

able

1S).

a Loc

atio

nof

the

SSR

with

resp

ectt

oth

ege

ne.W

hen

itha

sno

tbee

npo

ssib

leto

dete

rmin

e,N

.P.i

sin

dica

ted.

b The

expe

cted

PCR

prod

ucts

ize

inba

sepa

irs

base

don

the

sequ

ence

from

whi

chea

chpr

imer

pair

isde

rive

d.c T

otal

num

ber

ofba

nds

iden

tifie

din

the

tota

lnum

ber

ofan

alyz

edcu

ltiva

rs.

d Num

ber

ofba

nds

iden

tifie

din

each

cult

ivar

.e A

mpl

ific

atio

nan

dpo

lym

orph

ism

inth

epa

rent

allin

esof

the

dipl

oid

Fra

gari

are

fere

nce

segr

egat

ing

popu

latio

n.W

hen

apr

imer

pair

ampl

ifie

da

prod

ucti

nan

yge

noty

pe,a

‘+’

sign

isin

dica

ted.

Ifa

prim

erpa

iral

sode

tect

edpo

lym

orph

ism

sa

‘+/+

’sig

nis

indi

cate

d.W

hen

prim

erpa

irs

did

nota

mpl

ify

inF

.ves

cabu

tam

plif

ied

apr

oduc

tin

F.b

ucha

rica

,a‘-

/+’

sign

isin

dica

ted.

f Am

plif

icat

ion

and

poly

mor

phis

min

Ros

aw

ichu

rana

(E15

)an

d‘B

lush

Noi

sett

e’(D

10).

g Am

plif

icat

ion

and

poly

mor

phis

min

Rub

usid

aeus

cult

ivar

s‘T

ulam

een’

(Rt)

and

‘Mir

az’

(Rm

).

Mol Breeding (2011) 27:137–156 147

123

them consisting of dinucleotide and two of trinucleo-

tide repeats. A positive correlation was found when

the number of observed alleles was analyzed in

relation to the number of repeat units (r = 0.20,

p \ 0.05). When the correlation was investigated

separately for di- and trinucleotide repeats, a positive

correlation was still found for trinucleotides (r =

0.32, p \ 0.01) but not for di-nucleotide repeats

(r = 0.08, p = 0.57).

Transferability between Fragaria species

and mapping in diploid Fragaria

In order to evaluate the polymorphism in the diploid

Fragaria reference population, the developed primer

pairs were used to genotype the F. vesca 815 parental

line and the F1 hybrid. Among the 100 F. 9 anan-

assa newly developed SSRs, 90% amplified in

F. vesca fragments in the same range of size as that

in F. 9 ananassa. Therefore, ten strawberry primer

pairs failed to amplify in F. vesca 815, suggesting

that either these sequences have diverged between

both species or are not present in F. vesca. Consistent

with the idea that F. vesca contributes to the

F. 9 ananassa genome, all 22 F. vesca SSR markers

(100%) amplified in F. 9 ananassa. Strikingly, the

F. vesca SSR ChFvM125 did not amplify in F. vesca

815, suggesting the presence of a null allele in this

accession. We further analyzed the ten strawberry

SSR markers failing to amplify in F. vesca 815 in

four other accessions of F. vesca and two accessions

of F. iinumae. These were F. vesca 14 from Huesca,

Spain, F. vesca 184, F. vesca Reine des vallees 660,

the white-fruited F. vesca 596 and two F. iinumae

accessions (Table 1). In order to discard technical

problems and to be certain about primer specificity,

PCR amplifications were tested as least twice for all

accessions. Three markers (ChFaM035, ChFaM138

and ChFaM170) did not amplify in any of the

F. vesca or F. iinumae accessions, suggesting that

they are specific for F. 9 ananassa. Markers

ChFaM030, ChFaM148 and ChFv125 amplified

some accessions of both F. vesca and F. iinumae.

Markers ChFaM022 and ChFaM069 were specific for

F. vesca, failing to amplify any of the F. iinumae

accessions, while markers ChFaM047 and ChFaM70

specifically amplified one or both accessions of

F. iinumae (Supplementary Table 2S). Therefore,

our results indicate that two markers might be

specific to the Y1 genomic pool, or derived from an

F. vesca ancestor, while other two are Z genome

specific, or common to F. iinumae.

Out of the 122 primer pairs developed here, nine did

not amplify in either F. vesca 815 or F. bucharica 601

(Table 2). Among the remaining markers, 63 primer

pairs (56%) revealed polymorphisms and were ana-

lyzed in the bin set. In addition, five public but

unmapped SSR markers were also mapped in this

study (Materials and Methods). A total of 72 loci were

mapped in the diploid Fragaria reference map. Sixty-

three markers amplify one locus and five markers,

ChFaM017, ChFaM076, ChFaM140, ChFaM164 and

Fvi9, amplify two loci (labelled as a and b). In this

case, one (ChFaM076) or both (others) were bin-

mapped (Fig. 1). Two markers, ChFaM010 and

ChFaM114, did not correspond to identified bins and

were mapped in the whole FV 9 FB mapping popu-

lation. ChFaM010 defined a new mapping bin in

linkage group VII:68 and ChFaM114 defined a new

bin in linkage group III:20. To verify the segregation

of ChFaM017a, this marker was also scored in the

entire population mapping at position IV:26. Also, the

dominant marker ChFvM125 was scored in the entire

population mapping in position VI:87, cosegregating

with marker EMFv010.

The FV 9 FB bin map is at present composed of 49

bins, spanning 604 cM and with an average of seven

bins per linkage group (Fig. 1). The 72 new loci were

distributed across the seven linkage groups. Fourteen

markers were assigned to four bins on linkage group I,

12 markers to five bins in linkage group II, 12 markers

to five bins in linkage group III, 11 markers to three

bins in linkage group IV, six markers to four bins in

linkage group V, 12 markers to five bins in linkage

group VI and four markers to three bins in linkage

group VII. The two new mapping bins, defined by

markers ChFaM114 and ChFaM010, were localized in

linkage groups III and VII, respectively. Both markers

fill regions of the Fragaria reference map with a

reduced number of markers, dividing gaps of 16 cM in

two bins of 8 cM for ChFaM114 and 26 cM to 6 and

20 cM for ChFaM010.

Transferability of Fragaria SSRs to other

Rosoideae species

The genic-SSRs developed in this study together

with other 52 published SSRs (Table 4) were

148 Mol Breeding (2011) 27:137–156

123

Table 4 Other Fragaria SSR markers analyzed for transferability to Rosa and Rubus

Locus Reference Fragaria Rosa Rubus

Genic-–SSRs F. 9 a. cv.a Size range FV 9 FB E15/D10 Size range Rt/Rm Size range

ARSFL_31 Lewers et al. (2005) ?/? ?/? -/- – -/- –

ARSFL_35* Lewers et al. (2005) ?/? 210–212 ?/? ?/? 185–195 ?/? 210–212


ChFaM001 Gil–Ariza et al. (2006) ?/? ?/? -/- – -/- –

ChFaM002 Gil-Ariza et al. (2006) ?/? ?/? -/- – -/- –





ChFaM014 Gil-Ariza et al. (2006) ?/? ± -/- – -/- –

ChFaM021 Gil-Ariza et al. (2006) ?/? -/- -/- – -/- –


ChFaM029* Gil-Ariza et al. (2006) ?/? 147–182 ?/? ?/? 170–180 ± 184

FAC-003a Lewers et al. (2005) ?/? ?/? -/- – -/- –

FAC-005b Lewers et al. (2005) ?/? ?/? -/- – -/- –

FAC-006a Lewers et al. (2005) ?/? -/- -/- – -/- –

PBCESSRFXA12* Keniry et al. (2006) ?/? 180–213 ?/? ± 172 ± 176

UFFa01E03 Bassil et al. (2006a) ?/? ?/? -/- – -/- –

UFFa01H05 Bassil et al. (2006a) ?/? 250–290 ?/? ?/? 230–240 -/- –

UFFa02H04 Bassil et al. (2006a) ?/? ?/? -/- – -/- –

UFFa03B05 Bassil et al. (2006a) ?/? ?/? -/- – -/- –

UFFa15H09 Bassil et al. (2006a) ?/? ?/? -/- – -/- –

UFFa16H07* Bassil et al. (2006a) ?/? 235–290 ?/? ± 240 ?/? 237–244

Genomic-SSRs

ARSFL_007* Lewers et al. (2005) ?/? 225–275 ?/? ?/? 218–220 ± 205


BFACT-002 Rousseau-Guetin et al. (2008) ?/? 133–253 ?/? ?/? 190–202 -/- –

BFACT-008 Rousseau-Guetin et al. (2008) ?/? ?/? -/- – -/- –

BFACT-029 Rousseau-Guetin et al.(2008) ?/? 220–240 ?/? -/- – ?/? 240–270

BFACT-036 Rousseau-Guetin et al. (2008) ?/? 106–178 ?/? ?/? 235–260 -/- –

BFACT-037 Rousseau-Guetin et al. (2008) ?/? -/- -/- – -/- –



CFVCT-003 Monfort et al. (2006) ?/? ?/? -/- – -/- –

CFVCT-005A Monfort et al. (2006) ?/? ?/? -/- – -/- –



EMFn049* Sargent et al. (2004) ?/? 176–216 ?/? ± 180 ?/? 220–282

EMFn160 Sargent et al. (2006) ?/? ?/? -/- – -/- –

EMFv008 James et al. (2003) ?/? ?/? -/- – -/- –

EMFv010 James et al. (2003) ± 234 ?/? -/- – ± 205–275

EMFv023 Hadonou et al. (2004) ?/? ?/? -/- – -/- –

EMFv027 Hadonou et al. (2004) ± ?/? -/- – -/- –

Mol Breeding (2011) 27:137–156 149

123

evaluated for cross-amplification in two other

important genera of the subfamily Rosoideae, rose

and raspberry. In order to gain insight into their

polymorphism, two species of Rosa (Blush Noisette

and Rosa wichurana) and two accessions of rasp-

berry (Rubus idaeus Tulameen, from Canada, and

Miraz, from Russia) were analyzed. Cross-amplifi-

cation in rose and raspberry, allele sizes and the

presence/absence of polymorphism are shown in

Tables 2–4.

Forty-seven of the 174 SSRs (27%) amplified a

product or products of the approximate size expected

for a homologous locus in the genus Rosa (summa-

rized in Table 5). The transferability of Fragaria

EST-SSRs was found to be significantly higher than

that for genomic-SSRs (28.7 vs. 19.4%), in agree-

ment with its location in more conserved regions of

the genome. As expected, due to their phylogenetic

relationship (Potter et al. 2007), a lower transferabil-

ity was obtained for Rubus, as only 34 of the SSRs

(19.5%) amplified in at least one of the accessions

(Table 5). Once more, transferability of EST-SSRs

(20.3%) was higher than that of genomic-SSRs

(16.1%). The level of polymorphism of Fragaria

SSRs in Rosa and Rubus was high even though only

two accessions were analyzed for each genus, with

63.8 and 41.2% polymorphic SSRs for rose and

raspberry, respectively (Table 5).

Among the Fragaria transferable SSRs, 33 mark-

ers were polymorphic in the Fragaria reference

population, in octoploid strawberry cultivars and

were transferable to rose. In contrast, only 16

Fragaria SSRs were found to be polymorphic in

the Fragaria reference population, in F. 9 ananassa

and were also transferable to raspberry. These

markers may provide anchor points for genome

comparisons between Fragaria and these two genera,

provided they are also polymorphic in available rose

or raspberry mapping populations. Of all the 174

markers analyzed, 14 mapped SSRs cross-amplified

in both Rose and Rubus (denoted in Tables 2–4).

Because they are distributed across the seven linkage

groups of the Fragaria map (Fig. 1 and Sargent et al.

2007), they represent anchor markers for genome

comparisons among the three Rosoideae genera.

Discussion

The main goal of this study was to increase the

number of functional SSR markers developed in

Fragaria. About 190 non-duplicated SSR markers

Table 4 continued

Locus Reference Fragaria Rosa Rubus

Genic-–SSRs F. 9 a. cv.a Size range FV 9 FB E15/D10 Size range Rt/Rm Size range

EMFv029 Hadonou et al. (2004) ?/? ?/? -/- – -/- –

EMFvi018 Sargent et al. (2003) ?/? ?/? -/- – -/- –

EMFvi108* Sargent et al. (2003) ?/? 180–210 ?/? ± 198 ± 190

EMFvi136 Sargent et al. (2003) ?/? ?/? -/- – -/- –

FAC-004d Lewers et al. (2005) ?/? ?/? -/- – -/- –

Fvi9 Ashley et al. (2003) ?/? ?/? -/- – -/- –

Fvi20 Ashley et al. (2003) ?/? ?/? -/- – -/- –

UDF002 Cipriani and Testolin (2004) ?/? ?/? -/- – -/- –

UDF004 Cipriani and Testolin (2004) ?/? 125–154 ?/? ± 150 -/- –

UDF005 Cipriani and Testolin (2004) ?/? ?/? -/- – -/- –

* Transferable markers across the Rosoideae genera mapped in Fragariaa Amplification and polymorphism in F. 9 ananassa (F. 9 a.) cultivarsb Size range of PCR bands are only shown for F. 9 a. cultivars when primer pairs cross-amplified in Rosa or Rubusc Amplification and polymorphism in the parental lines of the diploid Fragaria reference segregating population. When a primer pair

amplified a product in any genotype, a ‘?’ sign is indicated. If a primer pair also detected polymorphisms a ‘?/?’ sign is indicatedd Amplification and polymorphism in Rosa wichurana (E15) and Blush Noisette (D10)e Amplification and polymorphism in Rubus idaeus cultivars Tulameen (Rt) and Miraz (Rm)

150 Mol Breeding (2011) 27:137–156

123

have been published to date from this species (Bassil

et al. 2006a; Gil-Ariza et al. 2006; Keniry et al. 2006;

Lewers et al. 2005; Monfort et al. 2006; Rousseau-

Gueutin et al. 2008; Sargent et al. 2008). With the

novel 100 F. 9 ananassa genic-SSRs reported here,

we have increased the number of available markers

by more than 50%. In addition, we have developed 22

novel genic-SSRs from F. vesca, which is one of the

diploid ancestors of this species. More than 92% of

the new markers were polymorphic among straw-

berry cultivars even though they originate from the

transcribed region of the genome and in *20% of

them the SSR was located in the ORF. It has been

reported that markers derived from transcribed

sequences may be less polymorphic but more robust

compared with genomic-SSRs (Varshney et al. 2005).

The genic-SSR markers developed here appear to be

as polymorphic as genomic-SSRs based on the

Table 5 Transferability of Fragaria SSR markers to Rosa and Rubus

Type of markers Number of markers Amplification % Polymorphism %

Cross-amplification Fragaria–Rosa

EST-SSRs 143 41 28.7 27 65.9

Genomic-SSRs 31 6 19.4 3 50.0

Total 174 47 27.0 30 63.8

Cross-amplification Fragaria–Rubus

EST-SSRs 143 29 20.3 12 41.4

Genomic-SSRs 31 5 16.1 2 40.0

Total 174 34 19.5 14 41.2

Fig. 1 Bin map of diploid Fragaria showing the position of

the new genic-SSR markers. The vertical bars represent the

seven linkage groups divided into the 49 bins defined by the

joint genotype of the selected individuals (shown inside each

bin). The two new bins defined in this study are shown in

darker shading and marker names together with the exact

position in the map in bold. Dominant markers are depicted in

italics. Genetic distances are shown on the left, indicating the

position of the last marker included in the bin according to the

framework map

Mol Breeding (2011) 27:137–156 151

123

number of alleles identified, thus representing a

valuable tool for genetic characterization of

strawberry.

We found comparable levels of polymorphism

between di- and trinucleotide motifs among the

strawberry genic-SSRs. Although dinucleotide SSRs

usually have higher levels of polymorphism com-

pared to trinucleotide, Fernandez-Silva et al. (2008)

showed also similar levels of polymorphism for

melon EST-SSRs. It has been extensively reported

that the degree of polymorphism increases with the

length of the SSR. Here, we also found a correlation

between repeat number and polymorphism in the

strawberry genic-SSRs. However, this correlation

was not observed for the dinucleotide repeats ana-

lyzed separately. Other studies have also found no

consistent relationship between number of alleles and

number of repeats, either across all motifs or

individually, and it may become more variable for

an intermediate level of repeats (Ferguson et al. 2004;

Fernandez-Silva et al. 2008), although the cause for

this remains elusive.

Based on homology analyses, functions were

assigned for more than 67% of the ESTs from where

the markers were developed, whereas a further 10%

were derived from sequences with similarity to

unknown proteins. About 23% of the ESTs did not

contain significant similarity to any protein, either

because of the limited sequence available or because

they are specific to strawberry. Since all of them can

be considered functional markers, they are candidates

for quantitative trait loci and can be associated to

phenotypic or adaptive variation of strawberry fruit.

Examples of this are the markers ChFaM079,

ChFaM126 and ChFvM210, which may be related

to hormonal responses during fruit growth and

ripening, as they show homology to proteins induced

by auxin or abscisic acid (Tables 2 and 3).

All SSRs derived from F. vesca cross-amplified in

F. 9 ananassa and therefore their transferability was

100%. Other studies have also shown high transfer-

ability levels between F. vesca and F. 9 ananassa,

ranging from 94 to 100% (Cipriani and Testolin

2004; Hadonou et al. 2004; Monfort et al. 2006).

These results are in agreement with an ancestor of F.

vesca contributing to the genome of F. 9 ananassa

(Rousseau-Gueutin et al. 2009). In general, it has

been reported that rapid genomic changes occur after

polyploidization (Chen 2007). The most frequent

modification is the loss of homoeologous copies of

many duplicated genes, retaining at least one copy in

one homoeologue genome. However, such a high

level of cross-amplification of F. vesca sequences

suggests that these changes have not been dramatic in

F. 9 ananassa. This is supported by the high colin-

earity recently found between these genomes, indi-

cating that major rearrangements have not occurred

(Rousseau-Gueutin et al. 2008; Sargent et al. 2009a).

The transferability of F. 9 ananassa SSRs to F. vesca

was only 89.1%, similar to the level of cross-

amplification (89%) reported by Bassil et al. (2006a).

The failure of SSRs derived from F. 9 ananassa to

amplify in F. vesca may be due to divergence in those

sequences between the species. This appears unlikely

since a similar cross-amplification level would be

expected for the F. vesca SSRs in F. 9 ananassa. A

more plausible explanation is that these markers

specifically amplify strawberry homoeologues con-

tributed by another donor after hybridization. Inter-

estingly, out of the five strawberry SSRs failing to

amplify in all F. vesca accessions tested, two markers

(ChFaM047 and ChFaM070) amplified fragments of

the expected size in F. iinumae. Two other markers

(ChFaM022 and ChFaM069) were specific to some

F. vesca accessions. Ongoing mapping studies in

F. 9 ananassa may reveal whether these SSRs are

specific markers for the Z and Y1 genomic pools of

the octoploid Fragaria genome.

The bin mapping of 72 genic-SSR markers has

significantly increased the overall number of markers

in the diploid Fragaria reference map to about 420

molecular loci (Sargent et al. 2008, 2009b). The SSRs

mapped in this study were, in general, assigned to

defined FV 9 FB bins. However, two markers,

ChFaM010 and ChFaM114, defined new bins in

linkage groups VII:68 and III:20 respectively, cover-

ing regions with a reduced density of markers.

Additionally, a number of SSRs mapped in this study

were localized to bins with medium to low marker

density, significantly increasing map saturation. The

use of this bin mapping strategy allows the efficient

and cost-effective localization of markers in genetic

maps, which in turn may help in the validation of

candidate genes located in the vicinity of qualitative

or quantitative traits. As an example, the marker

ChFvM125, identified in the fosmid EU024861, is

located at about 10 kb distance from FaCO, a

homologue of the Arabidopsis gene CONSTANS

152 Mol Breeding (2011) 27:137–156

123

(CO) which participates in the photoperiodic control

of floral induction (Putterill et al. 1995). The

SEASONAL FLOWERING LOCUS (SFL) regulates

seasonal flowering in the wild diploid F. vesca, which

flowers after short day induction. The position of SFL

has been reported at 29.6 cM on linkage group VI

(Albani et al. 2004; Cekic et al. 2001; Sargent et al.

2004), indicating that FaCO is not the gene respon-

sible for the seasonal flowering in F. vesca. Similarly,

our results also excluded a TFL1-like gene (marker

ChFaM142), mapped in bin V:48, as being the SFL

locus (Fig. 1).

Many studies have addressed the cross-amplifica-

tion of SSR markers within families. As examples, a

transferability of about 35% has been reported from

tomato to pepper (Frary et al. 2005) and up to 60%

among cereals such as rice, wheat, barley and maize

(Gupta et al. 2003; Tang et al. 2006). The transfer-

ability within the Rosaceae family has been shown to

be lower (Decroocq et al. 2003; Gasic et al. 2009).

Recently, a close phylogenetic relationship between

Fragaria and Rosa has been reported while the genus

Rubus, also belonging to the Rosodae supertribe, is not

as closely related (Potter et al. 2007). In accordance

with their phylogenetic distances, the transferability of

Fragaria SSRs was 27 and 19.5% to Rosa and Rubus,

respectively. This study has also shown that cross-

amplification of genic-SSRs was significantly higher

than that of genomic-SSRs: 28.7 vs. 19.4% for Rosa

and 20.3 vs. 16.1% for Rubus. Transferability of Malus

EST-SSRs to Pyrus, Fragaria, Rosa and Prunus has

been reported recently (Gasic et al. 2009). The highest

transferability, 62%, was found to the closely related

pear, followed by Prunus, with 56%. Although Malus

is distantly related to Fragaria and Rose, transferabil-

ity to these genera was high, with 48 and 28%,

respectively. In accordance with our results, low rates

of SSR cross-amplification have been reported

between Rosoideae genera, with transferability rates

of Fragaria SSRs to Rubus of between 27 to 19%

(Lewers et al. 2005). In contrast, these authors found

that none of the 30 Rosa-derived SSRs amplified

products in Fragaria or Rubus. In a recent report, a low

transferability of genomic SSRs from peach and apple

to rose was found: 17 and 8%, respectively (Oyant

et al. 2008). In contrast to the results reported here,

cross-amplification of Fragaria SSRs in rose was very

high (76%), although only 17 SSRs were evaluated.

Overall, these results suggest that sequence

conservation is higher among members of the Spirae-

oideae, such as between Prunus and Malus, than

among the Rosoideae subfamily.

We have found some inconsistencies between our

results and previously published data (Lewers et al.

2005). Thus, in the case of marker ARSFL_35, which

is located in the cellulase gene Cel2, we obtained a

clear cross-amplification in both raspberry accessions

tested, while it did not amplify in the study of Lewers

et al. (2005). This genic-marker might be a valuable

tool for genome comparisons since it is polymorphic

in all tested genera within the Rosoideae. Also,

marker EMFv010 did not amplify any of the tested

raspberry accessions in the study of Lewers et al.

(2005) but amplified one accession in our study,

whereas marker Fvi20 cross-amplified in their work

but not in ours. These differences might be due to the

different accessions tested, besides the wide diversity

reported within the Rubus genera (Jennings 1988).

Finally, an important issue in cross-amplification

evaluation is the PCR conditions used, which have

been maintained quite stringently in our study.

Lowering the Ta in the reaction may increase

transferability, but false positives can appear and

will produce incorrectly scored loci and errors in

genome comparisons.

Besides cross-amplification, it is necessary that a

marker developed for one species can detect poly-

morphism at homologous loci in related genera.

Despite other markers such as conserved orthologous

sequences that have been shown to be more transfer-

able across taxa, SSRs are characterized by a high

level of polymorphism. In this study, we have shown

a high level of polymorphism of Fragaria SSRs in

both Rosa and Rubus, even though only two acces-

sions were evaluated for each genus. Therefore,

although SSR markers are less transferable across

taxa, the probability of polymorphism between map-

ping genotypes is expected to be higher. Furthermore,

since genic-SSRs are derived from transcribed genes,

they are more conserved and transferable than

anonymous sequence-derived markers. Our study

has shown that genic-SSRs are significantly more

transferable than SSRs obtained from anonymous

genomic sequences.

Acknowledgments This work was supported by grants

RTA2008-00029-00-00 (INIA, partly funded by FEDER,

European Union), AGR-03230 (CICE, Junta de Andalucıa)

Mol Breeding (2011) 27:137–156 153

123

and BIO2007-67509-C02-01 (MEC). The IFAPA Fragariagermplasm collection is funded by grant RFP2008-00009-00-

00 (INIA). At CRAG, this work was supported with funds of

the Spanish Ministry of Science and Innovation, project

RTA2007-00063-00-00 (INIA). Y.Z-F. was supported by a

fellowship and I.A. by a contract from INIA. We are gratefull

to E. Cruz-Rus for obtaining the genomic sequence of GaLUR.

Ethical standards The experiments of this study comply

with the current laws of Spain.

Conflict of interest The authors declare that they have no

conflict of interest.

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Development and bin mapping of strawberry genic-SSRs in diploid Fragaria and their transferability...

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