Development, Characterization, and Transferability To Other Solanaceae of Microsatellite Markers in Pepper (Capsicum Annuum L.)

668
Development, characterization, and transferability

to other Solanaceae of microsatellite markers in
pepper (Capsicum annuum L.)
Istvan Nagy, Aniko Stagel, Zsuzsanna Sasvari, Marion Roder, and Martin Ganal
Abstract: A novel set of informative microsatellite markers for pepper (Capsicum annuum L.) is provided. Screening of
approximately 168 000 genomic clones and 23 174 public database entries resulted in a total of 411 microsatellite-containing
sequences that could be used for primer design and functional testing. A set of 154 microsatellite markers originated
from short-insert genomic libraries and 257 markers originated from database sequences. Of those markers, 147 (61
from genomic libraries and 86 from database sequences) showed specific and scoreable amplification products and detected polymorphisms between at least 2 of the 33 lines of a test panel consisting of cultivated and wild Capsicum
genotypes. These informative markers were subsequently surveyed for allelic variation and information content. The
usefulness of the new markers for diversity and taxonomic studies was demonstrated by the construction of consistent
phylogenetic trees based on the microsatellite polymorphisms. Conservation of a subset of microsatellite loci in pepper, tomato, and potato was proven by cross-species amplification and sequence comparisons. For several informative
pepper microsatellite markers, homologous expressed sequence tag (EST) counterparts could be identified in these related species that also carry microsatellite motifs. Such orthologs can potentially be used as reference markers and
common anchoring points on the genetic maps of different solanaceous species.
Key words: pepper, Capsicum spp., microsatellite (SSR) markers, diversity studies, polymorphism, cross-species transferability.
Resume : Une nouvelle collection de microsatellites informatifs chez le poivron (Capsicum annuum L.) est decrite.
Le criblage denviron 168 000 clones genomiques et 23 174 entrees dans les banques de donnees publiques a permis
didentifier 411 sequences contenant des microsatellites, de concevoir des amorces et de tester leur fonctionnement.
Un jeu de 154 marqueurs microsatellites a ete derive de banques genomiques a` inserts de petite taille alors que 257
ont ete obtenus de la banque de donnees. De ce nombre, 147 marqueurs (61 des banques genomiques et 86 des banques de donnees) ont produit des amplicons specifiques et lisibles et ont permis de detecter du polymorphisme entre au
moins deux dun panel de 33 lignees comprenant des genotypes cultives et sauvages du genre Capsicum. Lutilite de
ces nouveaux marqueurs pour des etudes de diversite et de taxonomie a ete demontree en produisant des arbres phylogenetiques concordants bases sur le polymorphisme des microsatellites. La conservation dun sous-ensemble des locus
microsatellite chez le poivron, la tomate et la pomme de terre a ete montree par amplification interspecifique et la
comparaison des sequences. Pour plusieurs microsatellites informatifs du poivron, des etiquettes de sequence exprimee
( EST ) homologues comprenant des microsatellites ont ete identifiees chez ces espe`ces apparentees. De tels orthologues peuvent potentiellement servir de marqueurs de reference et de points dancrage sur les cartes genetiques des differentes espe`ces de solanacees.
Mots-cles : poivron, Capsicum spp., marqueurs microsatellites ( SSR ), etudes de la diversite, polymorphisme, transportabilite interspecifique.
[Traduit par la Redaction]
Introduction
Microsatellites or simple sequence repeats (SSRs) are
DNA sequences consisting of tandemly repeated arrays of
short (16 nucleotides) motifs that frequently exhibit poly-
morphism between closely related genotypes. This polymorphism is probably due to slipped-strand mispairing, and
simple tandem repeats may be predisposed to further length
changes by unequal crossing over and (or) subsequent replication, repair, or recombination errors (Levinson and Gut-
Received 4 December 2006. Accepted 12 June 2007. Published on the NRC Research Press Web site at genome.nrc.ca on 3 August
2007.
Corresponding Editor: P. Gustafson.
I. Nagy,1 A. Stagel, and Z. Sasvari. Agricultural Biotechnology Center, Szent-Gyorgyi Albert u. 4, H-2100 Godollo, Hungary.
M. Roder and M. Ganal.2 Institut fur Pflanzengenetik und Kulturpflanzenforschung, Corrensstrae 3, D-06466 Gatersleben, Germany.
1Corresponding
2Present
author (e-mail: inagy1@gmx.net).

address: TraitGenetics GmbH, Am Schwabeplan 1b, D-06466 Gatersleben, Germany.
Genome 50: 668688 (2007)
doi:10.1139/G07-047
2007 NRC Canada
Nagy et al.
man 1987). As a result, microsatellite loci often mutate by

insertions or deletions of one or more repeat elements, and
the mutation rates generally increase with an increase in the
length of the repeats (Wierdl et al. 1997; Xu et al. 2000). As
locus-specific codominant markers that can be analyzed by
high-throughput PCR-based techniques, microsatellites are
ideal tools for different types of fingerprinting and genotype
identification tasks as well as for construction of framework
maps and for marker-assisted breeding (Rafalski et al.
1996). The benefits of using informative microsatellite
markers for saturation and integration of existing genetic
maps have been demonstrated in a number of plant species
such as wheat (Roder et al. 1995, 1998; Gupta et al. 2002),
rice (Temnykh et al. 2000), barley (Pillen et al. 2000; Ramsay
et al. 2000; Li et al. 2003), maize (Sharopova et al. 2002),
Cucumis spp. (Gonzalo et al. 2005), and tomato (Broun
and Tanksley 1996; Areshchenkova and Ganal 1999, 2002;
Frary et al. 2005).
Pepper (Capsicum annuum L.) is an important vegetable
crop worldwide. Besides having relatively large genomes
(3753 to 4763 Mb in different Capsicum species, Bennett
and Leitch 2005), cultivated pepper genotypes also exhibit a
low level of molecular polymorphism; the lack of hypervariable molecular markers in pepper hampers the construction
of saturated genetic maps. Although activities directed toward
microsatellite marker development and microsatellite mapping in pepper were reported recently (Huang et al. 2000;
Lee et al. 2004; Ogundiwin et al. 2005), and proprietary
microsatellite markers have been made available for diversity studies and genetic mapping (Tam et al. 2005; Ben
Chaim 2005), the number of presently available pepper microsatellite markers is far from the number that would be
necessary for high-density mapping and efficient gene targeting.
In this paper we report the development and primary characterization of a novel set of pepper microsatellite markers
obtained from genomic libraries and publicly available database sequences as well as data concerning the transferability of these markers to other solanaceous species.
Materials and methods

Library screenings
Genomic DNA from the pepper cultivars C. annuum Feherozon and C. annuum Blondy was digested with the restriction enzyme MboI or Sau3AI. Size-selected fragments
between 400 and 1000 bp were ligated into the BamHI site
of the ZAP Express1 vector (Stratagene). Packaging into
phages, plating, hybridization, and in vivo excision were
performed according to Roder et al. (1995, 1998).
The construction and management of plasmid libraries
enriched in single-copy sequences and the preparation of
high-density hybridization filters using the BioPick/BioGrid
robotic system (BioRobotics Ltd., UK) were carried out as
described by Pestsova et al. (2000). Filters were subsequently hybridized with 32P-labeled synthetic oligonucleotide probes representing the following motifs: poly(GA)
and poly(GT) for dinucleotide motifs; (ACA)8, (AGA)8,
(GAC)6, (ACT)7, (CAC)6, (GAG)6, (CGC)6, and (TAT)10 for
trinucleotide motifs; and (AACT)4, (ACAT)4, (GATA)4,
(GACA)3, (GGCC)3, and (GGTT)4 for tetranucleotide motifs.
669
Plasmid clones were sequenced on an ABI PRISM1 377 or

3100 automatic sequencer (Applied Biosystems) using
standard sequencing procedures.
Database searches
Nucleotide sequences belonging to the genus Capsicum
were retrieved from the EMBL database using the Sequence
Retrieval System (SRS) to build a stand-alone nucleotide database that could be searched using the utilities of the
BLAST2 package (Altschul et al. 1997). All further database
management and DNA sequence manipulations were carried
out on local PCs under the Linux operating system using
standard UNIX tools and public-domain bioinformatics software. Searches to identify entries containing short tandem
repeats were performed by the fuzznuc nucleic acid pattern
search program of the EMBOSS software package (Rice et
al. 2000).
Data processing
Microsatellite-containing clones were inspected individually with respect to sequence quality and suitability for PCR
amplification. Redundant clones were filtered out by local
BLAST analyses using the individual clones as queries
against the whole set of repeat-containing sequences. Primer
pairs flanking the microsatellite motifs were designed using
the Primer3 program (release 0.9, Rozen and Skaletsky 2000).
Plant material
To measure the level of microsatellite polymorphism, a
set of 33 pepper genotypes was used, consisting of cultivated
varieties, inbred lines, and wild Capsicum species.
For testing the cross-species transferability of the markers,
all developed microsatellite markers were tested for amplification in the following species of the family Solanaceae:
Solanum pimpinellifolium (formerly Lycopersicon pimpinellifolium), inbred line WaWa700; Solanum lycopersicum (formerly Lycopersicon esculentum), inbred line 4889; and
Solanum tuberosum Desiree (Table 1).
Analysis of microsatellite polymorphisms
DNA was isolated from leaves of greenhouse plants using
standard procedures (Dellaporta et al. 1983). PCR amplifications were carried out in 25 mL volumes with 1050 ng of
genomic DNA as template. Reaction mixtures contained
10 mmol/L TrisHCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/
L MgCl2, 0.5 U Taq polymerase, 200 nmol/L of the unlabeled
reverse primers, 160 nmol/L of the fluorescently labeled
forward primers, and 0.2 mmol/L of each deoxynucleotide.
An initial denaturation step at 94 8C for 3 min was typically followed by 35 cycles of 92 8C for 1 min, 60 8C for
1 min, and 72 8C for 1 min. The reactions were terminated
by a final extension step at 72 8C for 7 min.
For the first functional tests, unlabeled forward primers
were synthesized, and DNA of the parental and F1 genotypes of an interspecific (C. frutescens Tabasco C. annuum P4) and intraspecific (C. annuuum CM334 GF109)
hybrid combination was used as template. Amplification
products were run on agarose gels and (or) on ALFexpressTM II sequencers (GE Healthcare Life Sciences) with
the incorporation of Cy5-labeled dCTP (GE Healthcare
Life Sciences). In cases in which a new marker proved to
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Genome Vol. 50, 2007
Table 1. Genotypes used for polymorphism investigations.

Species
No.
Genotype
Inbred lines
Capsicum annuum
MK1 (sweet conical

wax)
MK2 (yellow blocky)
MK3 (tomato shape)
MK4 (white apple)
MK5 (Lamuyo type)
MK6 (red paprika)
MK7 (dark green
blocky)
MK8 (long pale green)
Yolo Wonder (green to
red blocky)
Perennial (chile)
P4 (red paprika)
GF109 (red paprika)
CM334 (chile)
MN1 (purple leaf ornamental)
2
3
4
5
6
7
8
9
10
11
12
13
14
Wild Capsicum genotypes
C. frutescens with
C. chinense introgression
C. chinense
C. eximium
C. pubescens
C. baccatum var. pendulum

C. praetermissum
C. chacoense
C. baccatum var. baccatum
C. frutescens
Solanum genotypes
Solanum pimpinellifolium
Solanum lycopersicum
Solanum tuberosum
15
Tabasco
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
chi1
chi7
chi21
chi25
exi1-1043
exi1-1052
pub2
pub3
pub5
pen1
pen6
pra1
cha6
cha9
bac10
bac20
fru39
fru48
34
35
36
WaWa700
4889
Desiree
be functional on this primary test panel (i.e., it produced

well-defined fragments and showed at least 1 polymorphism between the 4 parental genotypes), fluorescently labeled forward primers were synthesized to enable further
analyses on automatic sequencers. Fragment analysis of
PCR products labeled by the cyanine dye Cy5 using ALFexpressTM II sequencers was carried out as described elsewhere (Li et al. 2003).
To facilitate multiplexed semi-automated analysis of
larger numbers of samples on an Applied Biosystems 3100
capillary sequencer, forward primers labeled with FAM
(5-carboxyfluorescein) or JOE (6-carboxy-4,5-dichloro-
Table 2. Frequencies of microsatellite motifs in

pepper (Capsicum spp.) sequences in the EMBL
database and in genomic libraries.
Frequency*
Repeat
Database
(23 174 entries)
Genomic libraries
(55 296 clones)
A
C
AC
AG
AT
AAC
AAG
AAT
ACC
ACG
ACT
AGC
AGG
ATC
CCG
AAAC
AAAG
AAAT
AACT
AAGG
ACAT
ACAG
ATAG
AATT
AGGG
AAAAG
ACTAT
Total
181
8
17
104
29
21
60
20
37
2
7
24
12
27
15
0
2
3
0.4
0.4
0
0
0
0.4
0.4
0.4
0.4
571.4
n.a.
n.a.
12
11
10{
0.5
0.5
7
2
0
0.5
0.3
0.3
0.3
n.d.
0.3
n.d.
n.d.
0.6
n.d.
0.3
0.3
0.3
n.d.
n.d.
n.d.
n.d.
46.2
Note: n.a., not applicable; n.d., not determined.

*Occurrence per 10 000 clones or entries.
{
Not screened but found randomly.
2,7-dimethoxyfluorescein) were synthesized for selected

polymorphic markers. Amplification products were analysed after simultaneously running FAM- and JOE-labeled
PCR products and ROX (5-carboxy-X-rhodamine) labeled
internal size standards on 36 cm capillaries with POP4
polymer at 1500 V using a 15 s injection time. Fragment
analysis data from capillary runs were collected by the
built-in data collection software and pre-processed by the
GeneScan software (version 3.7, Applied Biosystems).
GeneScan data were imported, converted to pseudogel images, and further analyzed by the Genographer program
(Benham 2001).
Polymorphism information content (PIC) values were calculated according to the following equation (Anderson et al.
1992; Hedrick 1985):
PIC 1
k
X
Pi 2
i1
where Pi is the frequency of the ith allele and k is the total

number of different alleles at the locus. For each pepper
SSR marker, 2 different PIC indices were calculated: one
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Nagy et al.
671
Table 3. Functionality of the newly developed microsatellite markers on a test panel consisting
of 33 Capsicum genotypes.
Primer pairs tested

No amplification
Weak or unspecific products, not scoreable
Monomorphic
Functional markers
(PICa) for the information content in C. annuum (14 genotypes) and one (PICc) for all Capsicum genotypes from the
secondary test panel (33 genotypes belonging to 8 species,
including the 14 lines of C. annuum).
Cluster analysis
The presence or absence of each SSR allele was coded as
1 or 0, respectively. The resulting binary matrix was directly
used to construct unrooted phylogenetic trees with the DOLLOP program. Phenetic analysis was performed after calculating pairwise genetic similarity indices (Nei and Li 1979)
from the binary matrix, using the UPGMA method option
of the NEIGHBOR program. Consensus trees were reconstructed by the CONSENSE program; graphical rendering
of the dendrograms was processed by the DRAWTREE program (PHYLIP package, version 3.6, Felsenstein 2005).
Results and discussion

Genomic libraries
Approximately 60 000 phage clones and 108 000 plasmid
clones were used for the screening of microsatellite-containing
sequences. Based on an average insert size of 550 bp, 80%
of plasmids containing inserts, and 10% redundancy, the
total length of the screened genomic sequences was estimated
to be 66 000 kb. The screenings revealed 512 positive clones,
which corresponded to one positive clone for every 129 kb.
However, for most of the screenings only (GA)n and (GT)n
dinucleotide repeat probes were used for the plaque and
colony hybridizations, and a number of the positive clones
proved to be false positives after sequencing. To determine
more representative frequency estimates, 3 high-density filters
containing 55 296 colonies were sequentially probed with
di-, tri-, and tetra-nucleotide repeats. All positive clones
were isolated and sequenced and microsatellite-containing
clones from the test filters were considered for frequency
calculations.
Microsatellite motif frequencies
At the time of the survey, the EMBL sequence database
(release 76) contained 23 174 entries belonging to the genus
Capsicum. The majority of these entries (about 98%) represented short partial cDNA sequences (conventionally called
expressed sequence tags or ESTs). The total length of all
Capsicum sequences reached 10 828 kb. Search criteria
were set to identify microsatellite motifs of at least 16
mononucleotide, 8 dinucleotide, 5 trinucleotide, 5 tetranucleotide, or 5 pentanucleotide repeats, allowing for only a
single mismatch. The searches resulted in a total of 1322
microsatellites, of which 438 represented mononucleotide,
Genomic libraries
Database sequences
No.
154
29
40
24
61
No.
257
42
109
20
86
%
18.8
26.0
15.6
39.6
%
16.3
42.4
7.8
33.5
348 dinucleotide, 519 trinucleotide, 15 tetranucleotide, and

2 pentanucleotide repeats. Thirty-nine sequences contained
more than one microsatellite and 37 SSRs occurred in compound form (excluding mononucleotide stretches).
A/T mononucleotide repeats were the most frequent microsatellite motifs in the database sequences. To avoid targeting poly(A) tails of cDNA sequences, search criteria for
A/T mononucleotide repeats were set so that only sequences
that contained A/T repeats at least 20 nucleotides away from
either the 5 or the 3 end were selected. With such criteria,
in almost 2% of the database sequences, A or T repeats longer
than 16 bp were found. The frequency of C/G mononucleotide repeats was much lower (less than 0.01%). Screening
of genomic libraries for mononucleotide repeats was not
undertaken. The same was true for the (AT)n hybridization
probe owing to its self-complementarity. However, (AT)n
repeat motifs can be found relatively frequently at random
in the sequenced genomic clones, in the majority of cases
as part of compound repeats. The frequency of compound
SSRs is strikingly high in pepper genomic clones and,
most typically, (AC)n or (AG)n microsatellites are associated
with (AT)n repeats.
Trinucleotide repeats are the most abundant class of repeat
motifs in all cDNA-dominated sequence databases (Chen et
al. 2006; Cho et al. 2000; Thiel et al. 2003). In our study,
the frequency of all specific trinucleotide repeats was
39.2%, while the frequency of mononucleotide repeats was
33.1% and that of dinucleotide repeats was 26.3%, from
the total of 1322 identified microsatellites.
All 10 possible types of trinucleotide repeat motifs occurred in database sequences of pepper. The most frequent
repeat types in pepper were AAG, ACC, and ATC, while
ACG and ACT repeats were relatively rare. Similar frequency
patterns were found in the global plant sequence databases
(Katti et al. 2001; Toth et al. 2000), while CCG repeats
seemed to dominate monocot EST sequences (Cho et al.
2000; Thiel et al. 2003). The occurrence of trinucleotide
repeats was generally low in genomic libraries. Microsatellites with 5 or more repeat units of tetra- and penta-meric
repeats were found at very low frequency in both types of
sequence pools. The frequencies of different repeat types
found in genomic libraries and in database sequences are
given in Table 2.
To get some information on possible frequency overestimations, a redundancy analysis for database sequences was
performed for the most prominent dinucleotide repeat type,
(AG)n. Among 240 identified sequences containing (AG)n
repeats, 27 (11%) were found to be present redundantly,
with an average occurrence of 3.4 times. The overall redun#
2007 NRC Canada
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Table 4. Allelic variation and information content of the informative GPMS markers.
Repeat
(AC)18
GPMS3
(CA)17(TA)21
GPMS4
(GT)8GCG(TG)5(TA)7
GPMS6
(AC)20imp(AT)5
GPMS8
(AT)9(GT)22
GPMS29
(GT)15GGT7(GTT)2
GPMS37
(TC)18(TA)12
GPMS93
(TA)14imp(GA)27imp
GPMS100
T5(GT)12
10
GPMS101
(TC)16imp
11
GPMS103
(CT)11
12
GPMS104
(AT)6(GT)11
13
GPMS107
(AT)7(GT)12
14
GPMS109
(AC)14(AT)7
15
GPMS111
(AT)7(GT)12
16
GPMS112
(AT)8(GT)19
17
GPMS113
(AT)20(AG)18
18
GPMS117
(TA)25(GA)14
19
GPMS119
(GT)11G13
20
GPMS140
(TA)7imp(CA)7(TA)4
2007 NRC Canada
Forward primer / reverse primer (5?3)

CCCTAATGCTTGACGTGG/
GGTTAAGGGGGTTGGGG
ACTTGACAGTCGTGTATCTGCA/
GGACTCCACCAGCTCAGTTC
TTGATTTTTAAAGCAAAGTCGG/
ACAAGGAAATTCTTGCAGGG
CAGAGCACTTGACATGCCTT/
GATCTTTATAGTAGCTCATCAATA
TGATGATAAGGCCATGATAAAATG/
CCAGATTCTTTAGCAAGGTTTACC
CAGGCAATACGGAGCATC/
TGTGTTGCTTCTTGGACGAC
ATTTGTATATTATTTCTTGGCCTTG/
TGAACTACCCAATTCCAGCC
ATCCTTGGCGTATTTTGCAC/
TTCACTTTGCACACAGGCTT
TCCATACGGTTGGAGGAGAG/
ACTATGCTCTGCTGTGCCCT
CCTATCACCCTCTTTGAGCC/
TAAAGACCAGCCCTGGATGA
TGGCAGTTGCAATATCAATCTC/
AAACCTTGTCACGCATCCAT
GGGTCATGGGATTTCTTTTC/
ATTCAAACCATCCTTCAAGTTC
AACTAATTCTTGTCTGAGCA/
CATTTAACTTGATTTGATTG
ATCTATGCATGCCATCACCG/
TGGGCTAAAGGCCATGTTAC
TCAGAAGATGCCCATGTGTT/
TACTGGCACACGAAGCAATC
TCCCTCAGCAGCAACAATTT/
GTCGGGCTCTTTGATTGTGT
GCACAAGTCAATCCAAACGA/
CAAAAAGATGATGATGGATGAGA
GATGTTAGGTCCGTGCTTCG/
AAGCCCCATGGAAGTTATCC
CTGGAATCTGTCAATTGGTTG/
TCGTTTCATGATGGAATTGG
AACACATCGGTGGGTAATCC/
TGATTCCACCAGGCTTTAGG
Capsicum spp. (33 genotypes)
No. of alleles
4
PICa
0.49
No. of alleles
11
262282
0.73
196216
0.71
122172
0.68
12
0.89
159229
0.77
14
0.87
238255
0.60
0.83
176182
0.60
0.67
202268
0.44
12
0.86
141169
0.57
0.83
176211
0.46
0.67
134149
0.70
96123
0.13
0.72
177182
0.13
0.62
165183
0.13
0.48
120132
0.17
203280
0.76
10
0.86
91172
0.86
15
0.90
111177
0.76
11
0.86
204224
0.54
0.81
169199
0.63
PICc
0.83
Code
GPMS1
No.
1
Capsicum annuum (14 genotypes)

Allele size
range (bp)
121163
Repeat
(AC)7(CA)2(TA)5
22
GPMS142
(CA)6(TA)4
23
GPMS147
(CT)19
24
GPMS150
(AT)4(AC)9. . .(AT)33
25
GPMS151
(TC)13
26
GPMS153
(AC)10imp(AT)9
27
GPMS154
(CA)4A(CA)9
28
GPMS155
(TG)12impCA(TG)4
29
GPMS156
(GA)6(CA)6
30
GPMS157
(TG)7
31
GPMS159
(TAA)20
32
GPMS161
(AAT)25
33
GPMS162
(TA)2(TATG)4(TG)5
34
GPMS163
(AAC)5
35
GPMS164
(ACC)5
36
GPMS165
(TAA)35imp. . .(TA)26
37
GPMS166
(ACC)7
38
GPMS169
(ATT)5T(TTA)7
39
GPMS171
(TC)6. . .(TC)5. . .(TC)6
40
GPMS176
(TAT)13
41
GPMS178
(ATT)19
2007 NRC Canada

CATACATACACACGCGCATG/
TGAAATACGTTGTAATTATTATGGG
TTCGACCTCTTGGTATTCCG/
CACGACACATGGACTCCTTG
AGGAGGATATTTTGGCAGCC/
AAAAACACCCATGGGAAGTG
TGAGCGCTAACATATGTCGG/
GGCCCTTCCTCAGATTTCTC
ATCCTCGAACTTTGGTGTGG/
TGCATGCTCAGCAGGTAGAG
TGCTAGCTTCAACTGGTCCC/
TCAAATGTATGCATGCTTTGC
AATCTAATTTCTCGCCTGCG/
ATCACATTTTGGTTTTGGGG
GGGCGAATCGAGCTACATAG/
GGGATAGCGACAAAAAGTGC
GATATAGAACCACACAAGACTGGG/
TATGGCTCTTGCGGGAATAC
AATGCGCTGAAACCCAATAC/
TTTGAGGAAACACTAAAAAGTGTCC
AAGAACATGAGGAACTTTAACCATG/
TTCACCCTTCTCCGACTCC
CGAAATCCAATAAACGAGTGAAG/
CCTGTGTGAACAAGTTTTCAGG
TAACACACGCATGGTGAACC/
TCGGTTATCTTATACGGCCTG
CCACCGCTATCACTACCACC/
CCTCCGCAGTAGTGGTATGG
AATGAAATCAATCGGGCTTG/
ACCTCGCACCAATTCTTTTG
TGAACAATAATAATTGACAGGACAG/
AGCCTCGCAGTTTGTTCTTAC
AAAACCGACACACCAAAAGC/
CCCTAGTTTCCGTTGCAGAG
TCGAACAAATGGGTCATGTG/
GATGAGGGTCCTGTGCTACC
TCCACCACAATATTTCGAAGG/
TGGCTGTCCAACACTGTGAG
CCGCATTCTTGATTGAAACG/
TGGGCAGTATATTGTGCGTG
GATTTTTGACATGTCACATTCATG/
Allele size
range (bp)
100149
No. of alleles
1
PICa
0
No. of alleles
8
103125
0.56
184240
0.69
291299
0.58
183191
0.58
200225
0.26
165178
0.50
0.67
171173
0.13
0.06
228232
0.13
0.64
278305
0.68
281317
0.61
10
0.82
184259
0.8
12
0.89
305325
0.13
0.64
280291
0.57
230259
0.47
242317
0.61
13
0.87
193247
0.36
10
0.82
176220
0.52
0.78
288346
0.13
0.72
327345
0.13
0.70
230261
0.78
14
0.89
PICc
0.76
673
Code
GPMS141
No.
21
Nagy et al.
Table 4 (continued).
674
Table 4 (concluded).
2007 NRC Canada
Code
Repeat
42
GPMS181
(ATT)5. . .(ATT)11
43
GPMS183
(TC)9(TA)7
44
GPMS185
T12(GT)7
45
GPMS186
(TTA)14
46
GPMS187
(CA)6C4A36
47
GPMS188
(GGT)5(TGG)2
48
GPMS189
(AT)7imp(AC)7(AT)5
49
GPMS191
(GA)8
50
GPMS193
(CTT)8 . . .(CTT)10
51
GPMS194
(TA)17(GA)12
52
GPMS195
C11(CA)6
53
GPMS196
(TG)7(TA)6
54
GPMS197
(GA)3(TAT)16
55
GPMS198
T17(GT)9
56
GPMS199
(TA)16(GT)10(GA)16
57
GPMS200
(TC)6. . .(CT)4. . .(TC)7
58
GPMS201
(AC)4AT(AC)3
59
GPMS202
(AT)10. . .(AT)4(TG)9
60
GPMS203
(CAC)6
61
GPMS205
(AC)11(AT)5
Mean

AACGTTGAAAAATAAAGTAAGCAAG
GAAAGTGGAGAATTTAAAACCTTG/
TTCATTATCTCACCGTTACTGAC
GAGCTTCATAGATGATATGCAAGAG/
TCCCAAGCTAACCCATTTACTG
TCTCCCTGTAATTAGCAGAGCAG/
AAAAATCCCTAAATGAGCAAACC
GCGGTAAGGTCTGCGTACAC/
TTTGAAATAAGTTGGGCATAAATG
TTTAGAATCCTCACCACGGG/
TCAATGCACAAACTTTAATTTGC
TGCCGTGGTATTATTGGATG/
ATACCGACACACCAACCACC
GCTGCATCAAATCCCTAAGC/
TGATTCCACCAGGCTTTAGG
AGGTCAGCGACGGCAAC/
ATTTTAGGAGCCGACCTTCC
CGTACAATATCCATCCTTGTGC/
AAAAAGGAGAAAGATAAGGAAAAGG
AGGTGGCAGTTGAGGCTAAG/
GTTCTAGGTCTTTGCCCTGG
TGAAAACCAACCACTTGTGG/
TTGGAACTTACCGGAGTTGG
AAAGGATGCAACACGAGGAC/
TTCGCCTCACTGTGTTCTTG
GCAGAGAAAATAAAATTCTCGG/
CAATGGAAATTTCATCGACG
AGCTTTAGACAGTGTCTGCGTG/
TGATGATAAATTGCCTTCCG
TTGAAGTTGTGATTTTGCATG/
TTTCCGTAACAAATTTTCAAACC
CTGCGACTTAACGCTCACTG/
AATTAAACCATCGGGAACCC
ATCAACCCAGCAACACCAAC/
CTCCAGCAACAACAACTTCG
AAAGCTTTCAATAGTTTGGGGAC/
AGGGATAACGTGCAGAGCAC
CACCAACACATCTTTTTCAACC/
ATAATAGTGGTTGCGGCGAC
TCTCACTCCAAATTGGGGAG/
TGAACCAATATATTTTATACAAACC
Allele size
range (bp)
No. of alleles
PICa
No. of alleles
166214
0.13
0.64
195227
0.70
238242
0.25
0.71
161180
0.52
219246
0.71
121201
0.70
290321
0.68
261286
0.54
313335
0.65
216270
0.62
10
0.81
231321
10
0.77
159208
0.13
0.72
272344
0.81
10
0.82
252279
0.13
0.77
279371
12
0.74
233243
0.44
247250
0.17
252305
0.56
10
0.87
195210
0.68
313404
0.13
10
0.78
2.41
0.27
7.28
PICc
0.68
No.
Nagy et al.
Table 5. Allelic variation and information content of the informative EPMS markers.
Code
EPMS303
Repeat
(TA)25
EPMS305
(CTT)3(CAT)9
EPMS309
(CTT)6
EPMS310
(CAT)13
EPMS316
(AAT)8
EPMS327
(CGT)7
EPMS330
(AT)10
EPMS331
(CA)10
EPMS335
(ACAT)3(AT)17
10
EPMS340
(AT)13
11
EPMS342
(CTT)7
12
EPMS343
(CAT)6
13
EPMS345
(AAG)7
14
EPMS349
(GAT)6
15
EPMS350
(CAT)2(CAA)7
16
EPMS353
(CTG)6
17
EPMS366
(TA)8
18
EPMS369
(CAT)2. . .(CAT)6
19
EPMS372
(TA)8
20
EPMS373
(CAT)6

AAAACTCCAAACTACCCCTGG/
TTAAGCGTAGCGCTTGTGTG
CGTCTTTCACTTGTCTTTTGTTC/
AGTGGGTTCACTGACTTGGG
TCATCTTTCTGCAATTCCCC/
ACCAGGTTTAGCCCATGTTG
TGGGAAGAGAAATTGTGAAAGC/
AGGAAACATGGTTCAATGCC
CACCTCTCTAACCGTCACCC/
CAGAGAGCCAGGACAAGACC
TTTGGAACATTTCTTTGGGG/
ACGTAGCAGTAGGTTTGGCG
TGGGGACCAGATCCAGTTAC/
TTTCCGCCTATCAACAATGG
AACCCAATCCCCTTATCCAC/
GCATTAGCAGAAGCCATTTG
ATGCAGAGATTGTCGAAGCC/
GCAGAGAAGACTCACCAGTCC
AAACCTAGTGACTGGCGAGC/
AACGTTTTGCATAACGGAGG
CTGGTAGTTGCAAGAGTAGATCG/
ATGATCTTTGACGACGAGGG
TCTGGGAATTTTGGAACTGC/
TCCAGTTTTGATCATCTCCAAC
ATGGACGTTGATGAGCCTTC/
CGGCCTCCTGTTCTAATTTC
TGTAACCAAACTCGTGCTGG/
ATCAACCAATCAGCCTCGTC
AGTGTGGCTACGACAGCATG/
TTGTTTCCCTTACCTGCCAC
GTATGCTGCAACCATCGTTG/
ATTGGTTTGGGAGACACAGC
TTCGTTTTGTTCAGCTGAGAAG/
AACGATGAATTTGACGTCCC
TAATCGAGCGGTAGATTCGG/
TAAGTGGAGGTGCCCTTCTG
TCCACCCAGAAAAAGACTCG/
TGGACGGAACAAGAAATTTTG
TCTCCAATTTCCATTCGGAG/
Capsicum spp.
(33 genotypes)
No. of alleles
8
PICa
0.85
No. of alleles
9
94115
0.57
0.83
224250
0.07
0.70
140172
0.44
0.81
155204
0.13
0.31
98114
0.36
0.76
257268
0.13
0.67
92107
0.70
0.75
286330
0.66
10
0.84
261288
0.24
0.52
323343
0.50
0.78
129173
0.24
0.77
154163
0.50
223235
0.07
0.62
96106
0.3
0.75
299317
0.13
0.70
171196
0.73
347356
0.50
0.71
321325
0.24
0.60
177190
0.60
Allele size
range (bp)
291330
PICc
0.84
675
2007 NRC Canada
No.
1
Capsicum annuum
(14 genotypes)
676
Repeat
21
EPMS374
(CAA)6CAG(CAA)3
22
EPMS376
(CAA)6
23
EPMS377
(AG)11
24
EPMS378
(CGG)7(AGG)2
25
EPMS382
(ACA)9
26
EPMS386
(CA)15
27
EPMS387
(AAT)2AGT(AAT)6
28
EPMS390
(ATT)8
29
EPMS391
(AT)9
30
EPMS395
(CCG)6
31
EPMS396
(CAT)6
32
EPMS397
(CA)20
33
EPMS399
(AAT)8
34
EPMS402
(CCT)3(CTT)6(CCT)
35
EPMS404
(CTT)12
36
EPMS409
(CAT)7
37
EPMS410
(CT)14(CA)16 imp
38
EPMS411
(TA)9(GATA)3
39
EPMS412
(AT)9
40
EPMS413
(CCA)9
2007 NRC Canada

TAATCGCATTTGCGAACTTG
TTTTCCCCATGAATAAAGATGG/
TTCAATTTCACCTTCTGGGG
ACCCACCTTCATCAACAACC/
ATTTGTGGCTTTTCGAAACG
GACAGTCTTTCAAGAACTAGAGAGAG/
TGGAGCAAACACAGCAGAAC
CGAAGATCACCACCACACTG/
ACATTTTCACTGCCGGAGAC
TTTCCCCCATCACATGAAAG/
CCTAAAGCCATGCAAAAACC
ACGCCAAGAAAATCATCTCC/
CCATTGCTGAAGAAAATGGG
AGGGTTTCCAACTCTTCTTCC/
CTAACCCCACCAAGCAAAAC
GCTCCTCATTAGTAGCCCCC/
TCTTCATCACACCTCTGTGGAC
TTTCTTCTCTGGCCCTTTTG/
ACGCCTATTGCGAATTTCAG
TGTTGGAGCACCATACTCAAAC/
CATCTCCCGACTGAAACTCC
TCATCGTCTCTTCAACTCTTCTTG/
CCCAATTCGACCTGTTATGG
GCACCCTCCCAATACAAATC/
GATCACGGAGAAAGCAAAGG
GTTCTTCTCGCCGACAAGTC/
TTCAAAAATCATGAGCAGCG
GCCTTCTTTTTCATCTTTCCC/
CTGGCAACCCAAGTCTTAGC
TCTCTCTCTACATCTCTCCGTTG/
TGTCGTTCGTCGACGTACTC
ATACTAAGGCGTTTGGTCGG/
ACAATTGGGGATGCAGAAAG
GGAAACTAAACACACTTTCTCTCTC/
ACTGGACGCCAGTTTGATTC
AAAGGCAACGATGAGAATGG/
AGGACACAAGCACACATCATATC
CGGACTTTTCAAAAACACACAC/
ACCGACGGCAATGATCTTAC
GCCTCATAAAGGTGGCC/
Capsicum spp.
(33 genotypes)
Allele size
range (bp)
No. of alleles
PICa
No. of alleles
206215
0.69
235259
0.67
0.85
134161
0.50
0.72
101110
0.58
154166
0.70
122170
0.62
11
0.86
162199
0.5
10
0.80
113131
0.41
0.69
177187
0.5
0.79
116131
0.24
0.46
223231
0.57
0.67
102117
0.54
0.76
144175
0.37
0.81
197216
0.47
0.74
215245
0.47
0.80
162180
0.26
0.72
149187
0.54
10
0.85
333373
0.66
205213
0.49
0.77
252268
0.56
PICc
Code
No.
Capsicum annuum
(14 genotypes)
Nagy et al.
Repeat
41
EPMS414
(AT)10
42
EPMS415
(CCA)CA(CCA)7
43
EPMS416
(CTT)10T(CTT)2
44
EPMS417
(TC)9
45
EPMS418
(CA)10
46
EPMS419
(AAT)6
47
EPMS420
(CCG)9
48
EPMS421
(CCG)6
49
EPMS424
(CCT)6
50
EPMS426
(AT)15
51
EPMS427
(AAT)6(AAG)
52
EPMS428
(GAT)6
53
EPMS429
(ATC)8
54
EPMS430
(GAA)5ACA(GAA)
55
EPMS438
(ATT)4(TTA)2
56
EPMS439
(TC)6. . .(TC)7
57
EPMS440
(CT)7
58
EPMS441
(AG)11
59
EPMS443
(TG)13imp(TA)4
60
EPMS446
(CAA)AG(CAA)6
2007 NRC Canada

AGTTGGCGGTTTAACTGGTG
TTGGGGACTTCACGTCTCTC/
TTGATGATAAATCCTCCCCC
GTCGAACAAAATGGGGTTTG/
GCTGGAGAGTGCTGGTGG
GCCAAATGGAGGGTCCTTAG/
CAAAGAAAGATGAAGAAGCAACAG
CGCATATACATACATAAATTCTTTC/
TCAACATCTCACCGAAGCTG
ATCTTCTTCTCATTTCTCCCTTC/
TGCTCAGCATTAACGACGTC
TTCAGGTGCAGGTATCATCG/
GGGTACTTGTCCATTTATCCAG
GCATAATCGGAAAATGACGTG/
CAGCCAACTCCGAAAGCTAC
ATCTATTTTCCTCCGGCGAC/
CGGTAAGCTGCCTTGATCTC
TCTTTTCTCTCCACCCCATG/
TTGGGCATCTTTTTCAAAGG
GAGGAAACACTCTCTCTCTCTCTCTC/
TCAAGAGACCCCAAATAGGG
GCTCCTCATTAGTAGCCCCC/
TCTTCATCACACCTCTGTGGAC
GGCAACCCCAATGTTGTAAC/
CAACTCCAAACCCCTTAGGC
ATACTAAGGCGTTTGGTCGG/
ACAATTGGGGATGCAGAAAG
CCAACACCAAGAAATTGACG/
CCACTTGTGACCCATTAGGG
GCTTTTTGGCAGTTTCGTTC/
TTCCCCAGTTCACTCCTAGC
ACTAAAGGATCCCCCGGG/
CTCCCACCTGGACTCATCTG
ACGAGGCGCCCTCTCTC/
GAGTCCAAACTGAAGCTGCC
GCACGAGGAAAGAGAGAGACATAG/
TCAACGGATTCAGTCTTCCC
GGTTTTCTCACAACTTCGGC/
TTGCAAAATATATCAACGCG
GAGGCCTTCTGAACCAAACC/
Capsicum spp.
(33 genotypes)
Allele size
range (bp)
No. of alleles
PICa
No. of alleles
140186
0.07
0.77
212215
0.50
131154
0.36
0.79
110126
0.72
0.78
178210
0.84
12
0.88
224248
0.46
0.78
110116
0.55
236258
0.29
0.77
148160
0.53
108118
0.60
0.62
113131
0.41
0.75
318327
0.53
136180
0.26
0.66
255258
0.49
320332
0.46
246250
0.41
0.35
178192
0.49
116124
0.74
0.82
190196
0.34
0.58
328338
0.50
0.56
PICc
677
Code
No.
Capsicum annuum
(14 genotypes)
678
Repeat
61
EPMS448
(TAA)7
62
EPMS449
(TTA)7imp
63
EPMS451
(AT)9
64
EPMS472
T16
65
EPMS480
C12A7
66
EPMS490
T17
67
EPMS492
A7CA16
68
EPMS497
G14
69
EPMS501
T20
70
EPMS507
A30
71
EPMS514
A18
72
EPMS538
(CAT)4imp(CAC)2(CAT)3
73
EPMS539
(TC)11
74
EPMS540
(TGG)GG(TGG)5
75
EPMS542
(TC)10
76
EPMS543
(TC)4(CT)13
77
EPMS546
(ACC)7imp
78
EPMS547
(AG)7
79
EPMS549
(ACC)4TC(ACC)4
80
EPMS554
(TC)6T8
2007 NRC Canada

TTGTAAAGCCTTGGAGGTGG
GGGACGTATTTTCGAAGAGG/
CTTCGCCTTGTTGACTAGGG
GCGGAGCTCACAATAAGGAG/
TGACCCAATGCTCTTCTATGC
CGTAAGACTTTACAGTGAGAGAAATC/
ATTCCAGTGTCGCCGACTAC
ATTGTGATAGCAACCCCTGG/
CACAGATGAGGGCACAAATG
AGGAACGGCAGTCTTGCTAG/
GATGCTAGGTCTGGATTCCTG
TTGAGGAAACGTGTGCTGAG/
CGGCATTATTTCATAAGGTGG
CTCTCCAATCCTTCCCTTCC/
GAAGAAGAAGCTAGGTTTGTTTCG
TACACACACCATCGGGAAAG/
CAGTTTAGCCGAGTTTTCCG
AATCCTCCAAATCCACCCTC/
ATTCGATTGCTTGCTCCTTG
CGGGCAGGTGCTATTATAAAAC/
CGGCCGAGGTACAAGCC
GCCCATTATGACTGCACATG/
CACCAAATATTCCCTGAAAGG
AAGTTGCTAACGGTGCTTGG/
TGAAGTTTCAGGAGCTGCTTC
AGTGGGTTTCCTATTTGGGC/
CCATGAGCTGTGTTTGATGG
TGCTATTCCAGGTAAAGCCG/
TGTCGTGCGAGTCAGTCAG
ATCCACTTCCCCATTATCCC/
TGGATGATCGAGTTGACTGG
CTCTGCCCTCCTCAACCC/
AAAATATGGTCGGAGATCCG
AGATAATCATGGCGGCTCAG/
ATCTGATGATGCCACAGCTG
TCCTCCACTCAGCTTTCCTC/
TTTCCTTTTGATGAATCAGGATC
ACCAATGGATTGAATCCACG/
AGGGCAGTTGTAGCCACTTG
CATCATTTCTCCCCAATTCC/
Capsicum spp.
(33 genotypes)
Allele size
range (bp)
No. of alleles
PICa
No. of alleles
127161
0.76
134147
0.17
166184
0.36
0.80
289320
0.61
0.80
241255
0.69
0.69
216222
0.49
0.64
187202
0.46
236248
0.50
0.78
166180
0.69
10
0.88
196254
0.07
0.56
251267
0.13
0.73
118119
0.13
0.50
241254
0.46
0.80
188206
0.13
0.69
168228
0.68
10
0.89
101111
0.72
219230
0.46
0.68
95108
0.55
150160
0.58
209199
0.60
PICc
Code
No.
Capsicum annuum
(14 genotypes)
0.67
5.86
0.30
2.40
0.44
4
0
1
190208
0.75
4
0.49
2
167177
0.77
5
0.46
2
110117
0.33
2
0.13
2
118120
0.63
3
0
1
241243
3
0
1
183186
No. of alleles
PICa
EPMS563
86
Mean
(GA)9
EPMS562
85
(TC)11
EPMS561
84
(CT)4CC(CT)6
EPMS558
83
(CTT)5imp(CT)4
EPMS557
82
(CT)2T(CT)6
EPMS556
81
(CCT)5(CTT)2
Code
No.
Repeat

GTGGTGGGTGGGGTAAAAAG
TTTCTCATGTTGACTCCCAAG/
TCCATCTTTATCAGCTGGCC
TCTATTCTCCTCTCAAAACTTACGTC/
GAAACGAAGGAGGAAAAGGG
TCAACTCCATCAGTCTCCCC/
AACGCCTTGAATTAATTGCG
ATTGGACTTCAAATTTGGCC/
AAACCAAAATCAGCATTAAAATATAAAC
GGCCAACTCTCTCTCGTGTC/
TGGACAGGCGAATAGGGTAG
CTCATTACCACTTCATACAAAACAG/
TGCAGTAGGTGTTGCTACGG
Allele size
range (bp)
No. of alleles
Capsicum spp.
(33 genotypes)
Capsicum annuum
(14 genotypes)
0.17
679
PICc
Nagy et al.
dancy within this repeat class was calculated to be 1.37.

This leads to the conclusion that frequency estimates were
not substantially biased by redundancy for this repeat type.
While this estimation might not be precisely the same with
other repeats, data from other plants (Thiel et al. 2003) suggest no significant differences for other repeat types.
Analysis of microsatellite polymorphisms
After inspection of all positive microsatellite-containing
sequences, 411 could be chosen for primer design and functional testing. One hundred and fifty-four markers originated
from genomic libraries (GPMS markers) and 257 originated
from database sequences (EPMS markers). In all, 147
markers (61 from genomic libraries and 86 from database
sequences) showed specific and scoreable PCR products
and detected polymorphisms between at least 2 members of
the secondary test panel consisting of 33 Capsicum genotypes. (See Table 3 for the results of the functional tests.)
In the majority of cases, the PCR product size was in good
concordance with the predicted size. Five EPMS markers
(EPMS342, EPMS349, EPMS353, EPMS369, and EPMS413)
produced amplicons that were 70 to 100 bp larger than expected, probably because of the presence of introns.
EPMS490 produced fragments that were 50 bp shorter
than predicted. The repeat types, allele size ranges, and information contents of the informative GPMS and EPMS
markers are shown in Table 4 and Table 5, respectively.
(Primer data for the non-informative EPMS and GPMS
markers can be obtained from the authors upon request.)
Although GPMS markers detected more alleles (7.28
2.68, on average) than EPMS markers (5.86 2.00, on
average) in the full set of investigated Capsicum genotypes, the mean PICc values (based on allele-size variations
of 33 Capsicum genotypes) of the 2 marker classes were
similar (0.68 0.13 and 0.67 0.12, respectively). The selected informative markers showed a low level of polymorphism within C. annuum: with low mean intraspecific
(PICa) values (0.27 for GPMS markers and 0.30 for EPMS
markers), 41% of the GPMS markers and 28% of the
EPMS markers detected only one allele within the 14
C. annuum genotypes of the test panel (see Fig. 1B as an
example). On the other hand, some highly informative
markers exhibited relatively high allelic variations within
C. annuum (Fig. 1A). Correlation coefficient calculations
between PICc values and core repeat length resulted in r
values of 0.31 (P < 0.01) for trinucleotide repeat markers,
0.23 (P < 0.05) for dinucleotide repeat markers, and 0.61
(P < 0.1) for mononucleotide repeat markers, indicating
that there was no clear correlation between core repeat
length and informativeness. Similar observations have been
reported for other plant species such as maize (Chin et al.
1996), Cucumis spp. (Danin-Poleg et al. 2001), rice
(Panaud et al. 1995), and barley (Li et al. 2003; Struss
and Plieske 1998). These findings are discrepant with the
general assumption that an increased number of repeat
units is associated with a higher degree of polymorphism
(Weber 1990). In other studies, a clear positive correlation
was found between repeat length and informativeness, e.g.,
tomato (Areshchenkova and Ganal 1999; Frary et al. 2005;
He et al. 2003), rice (Cho et al. 2000), ryegrass (Jones et
al. 2001), and grapevine (Thomas and Scott 1993).
#
2007 NRC Canada
680
Fig. 1. Polymorphism of microsatellite markers EPMS386 (A) and EPMS382 (B). Pseudogel images were produced from data obtained with
an ABI PRISM1 3100 capillary sequencer. Genotypes and their order are as shown in Table 1.
Genetic diversity in Capsicum revealed by microsatellite

markers
A phylogenetic tree was constructed based on microsatellite
allele polymorphisms detected in 33 pepper genotypes using
147 informative microsatellite markers. The 33 genotypes
of the test panels belonged to 8 species of the genus Capsicum. The structure of the dendrogram was in good concordance with the taxonomical classifications: all C. annuum
genotypes located in the same main cluster divided into
sub-clusters that corresponded to fruit morphology characteristics (Fig. 2). The small-fruited exotic types (CM334,
Perennial, and MN1) make up a group that is clearly separated from the large-fruited cultivated varieties. The Hungarian red paprika genotypes were again separated from
the fresh market lines. The latter formed 2 subgroups: the
first contained the blocky lines (MK2, MK7, and Yolo
Wonder) and the Lamuyo line (MK5), while the second
one was a more morphologically heterogeneous subgroup

consisting of round, conical, and long-fruited lines (MK1,
MK3, MK4, and MK8). The main cluster nearest to the
C. annuum cluster contained the C. chinenese and C. frutescens genotypes. These 3 species are conventionally
grouped into a common taxonomical complex (Pickersgill
1988).
Tabasco is classified as C. frutescens, but in this dendrogram it is interspersed between the C. frutescens and
C. chinense subgroups. This separation may be explained
by the fact that the Tabasco line used in this study originates from the cultivar Greenleaf Tabasco, which was obtained from a cross of the traditional Tabasco and
C. chinense (Greenleaf et al. 1970). The third main cluster
included the remaining wild Capsicum genotypes of the
test panel. Lines of the 2 botanical varieties of C. baccatum
(C. baccatum var. baccatum and C. baccatum var. pendu#
2007 NRC Canada
Nagy et al.
681
Fig. 2. A phylogenetic tree of 33 Capsicum genotypes based on polymorphism of 147 informative microsatellite markers (see explanations
in the text).
lum) were located on 2 different forks of the same branch

and seemed to be more closely related to the C. chacoense
C. praetermissum subgroup than to the other neighboring
subgroup containing C. pubescens and C. eximium.
Besides the cladistic (parsimony) analysis, phenetic analysis
based on Neis similarity indices was also attempted (data
not shown). The topology of the dendrograms obtained by
the unweighted pair group method with arithmetic averages
(UPGMA) was nearly identical to that produced by the
parsimony analysis using the DOLLOP program, indicating
that the microsatellite polymorphism data allowed equally
plausible and largely congruent tree construction by means
of both cladistic and phenetic approaches.
Homology and cross-species transferability of the new
pepper microsatellite markers
All informative pepper markers were surveyed for amplification in the related species Solanum lycopersicon, Sola-
num pimpinellifolium, and Solanum tuberosum. In all, 44%

of the GPMS markers (27 markers) and 55% of the EPMS
markers (47 markers) produced detectable PCR products in
the related species. Of these 74 markers, 3 did not amplify
from S. pimpinellifolium, 6 amplified from potato and pepper but not from tomato species, 2 did not amplify from potato, and 63 amplified from all investigated species. When
detected on automated sequencers, the amplification products
were often weaker and contained more shadow bands in
the related species than in pepper (data not shown) but
could still be scored. To obtain more straightforward information on homology relationships of these newly developed pepper markers, local BLAST analyses were carried
out using all informative EPMS markers as queries. The
target database contained all available sequences of the
family Solanaceae of the dbEST (http://www.ncbi.nlm.nih.
gov/dbEST/) and EMBL databases (374 630 entries for this
family on the date of the analysis). Furthermore, the same
#
2007 NRC Canada
682
Table 6. Homology relationships of the informative EPMS markers.
No.
1
Code
EPMS303
Accession
No.
BD076366
EPMS305
Related pepper unigenes
EST homologs
Unigene
No.
Tomato
Potato
BI926274
CK278805
EPMS309
BM061028
KS01023E09 KS01 Capsicum annuum cDNA
U205213
EPMS310
BM061162
KS01027C08 KS01 Capsicum annuum cDNA
U199235
EPMS316
BM062268
KS01040F01 KS01 Capsicum annuum cDNA
U199490
EPMS327
BM063302
KS01053H01 KS01 Capsicum annuum cDNA
EPMS330
BM063625
U197030
U199197
U197581
EPMS331
BM063830
KS01060A10 KS01 Capsicum annuum cDNA
U199259
U196576
EPMS335
BM064021
KS01062B08 KS01 Capsicum annuum cDNA
U203480
10
11
EPMS340
EPMS342
BM064640
BM064842

U202270
U202848
12
EPMS343
BM064867
U199025
13
14
15
EPMS345
EPMS349
EPMS350
BM065407
BM066130
BM066247

KS07012D02 KS07 Capsicum annuum cDNA
U197842
U201952
U197448
16
EPMS353
BM067271
U196304
17
EPMS366
CA514758
U200913
18
19
20
21
EPMS369
EPMS372
EPMS373
EPMS374
CA515055
CA515633
CA515649
CA516096

U203361
U205108
U203547
22
EPMS376
CA516334
U198266
23
EPMS377
CA516439
U197562
24
EPMS378
CA516454
U199131
BLAST annotation
CAN131456 Capsicum annuum pap gene,
exons 13
Tetratricopeptide repeat (TPR)-containing protein (Arabidopsis thaliana)
Expressed protein T45782 hypothetical protein
(Arabidopsis thaliana)
Lactoylglutathione lyase family protein F8468
Wun1 wound-induced protein JQ0398
(Solanum tuberosum)
Fiber protein Fb19 (Gossypium barbadense)
AW040246
2007 NRC Canada
BQ508385*
BG599565*
AW441453
BQ506266
Late-embryogenesis protein lea5 T01984

(Nicotiana tabacum)
AI486006
BI435779
RuBisCO subunit binding-protein alpha subunit

Potato 13-lipoxygenase T07065 (Solanum
tuberosum)
Calcium-dependent protein kinase 3 (Capsicum

annuum)
C2 domain-containing protein G86222
Nucleolar protein (Cicer arietinum)
Polyprotein-related protein MDN11.13

Expressed protein At4g17940.1 68411.m02436
AP2 domain transcription factor (Arabidopsis
thaliana)
Putative metal ATPase (Arabidopsis thaliana)

Unknown protein NP_199491 (Arabidopsis
thaliana)
Expressed protein MHF15.9 (Arabidopsis
thaliana)
Unknown protein BAC79927 (Oryza sativa)
BW692801
BQ512574*
BQ506610*
BG642614
BM109649*
BG596055*
AW443210
BM404104
U204013
BM059622
Description of pepper source sequence

Capsanthin-capsorubin synthase gene promoter
(Capsicum annuum)
Nagy et al.
Code
Accession
No.
25
26
27
28
EPMS382
EPMS386
EPMS387
EPMS390
CA517071
CA517699
CA518417
CA519104

29
EPMS391
CA519548
30
31
EPMS395
EPMS396
CA520931
CA521318

32
33
34
EPMS397
EPMS399
EPMS402
CA521689
CA522816
CA523427
KS11031G02 KS11 Capsicum annuum cDNA

35
EPMS404
CA524065
36
EPMS409
CA525246
37
38
39
EPMS410
EPMS411
EPMS412
CA525390
CA525873
CA526181

40
EPMS413
CA526196
41
42
EPMS414
EPMS415
CA526211
CA514621

43
EPMS416
CA515656
44
45
EPMS417
EPMS418
CA514714
CA516044

46
EPMS419
CA523208
47
48
EPMS420
EPMS421
CA513842
CA514272

EST homologs
Tomato
Potato{
CK246344
AW622335
AI484478
BQ508386
AW647879
BQ517359
BE923711
AW617461
CK270861
AY423549{
BG096749{
BI203623
BM113158
AW039723*
AI775382*
BQ516501*
AI896317{
BI176058*
BQ512883{
683
2007 NRC Canada
No.

Unigene
No.
BLAST annotation
U199132
Peptidylprolyl isomerase-like protein
U202390
U205221
U202660
Glyoxalase/bleomycin resistance protein (Oryza
sativa)
U200675
Expressed protein At1g26948 (Arabidopsis
thaliana)
U198176
Imidazoleglycerolphosphate dehydratase
U196920
GTP-binding protein Rab6 (Nicotiana tabacum)
U203723
U204981
PAP2 AUX/IAA family phytochrome-associated
protein (Arabidopsis thaliana)
U204859
LRR-containing F-box protein AtFBL2
U198459
Thioredoxin family protein At2g35010
U201498
U198173
U198626
Isomerase-like protein AT4g15940/dl4011w
U196306
Proline-rich glycoprotein T01365 related
U198796
MADS-box protein 9 (Petunia hybrida)
U196307
U199066
U196306
Proline-rich glycoprotein T01365 related
U199271
Aux/IAA protein (Solanum tuberosum)
U199272
U196308
Fructokinase (Solanum lycopersicon)
U196436
Ethylene-responsive protein1 S60047 (Hevea
brasiliensis)
U198464
U196452
Translation-inhibitor protein (Gentiana triflora)
U198010
T11855 DnaJ protein homolog (Phaseolus
vulgaris)
684
2007 NRC Canada
Code
EPMS424
Accession
No.
CA514316

50
51
EPMS426
EPMS427
CA517376
CA519128

52
EPMS428
CA523880
53
EPMS429
CA525246
54
EPMS430
BM063052
55
EPMS438
CA847350
56
EPMS439
CA847439
57
EPMS440
CA847460
58
EPMS441
CA847465
59
EPMS443
CA847580
60
EPMS446
CA847612
61
62
63
EPMS448
EPMS449
EPMS451
CB164897
CB165039
CF270070
64
EPMS472
BM061910
EST0257 CM334 root cDNA, Capsicum

annuum cDNA clone RR61-05, similar to
Arabidopsis thaliana beta-1,3-glucanase-like
protein
Arabidopsis thaliana putative RNA-binding
protein; 2480823340 protein
Arabidopsis thaliana F2D10.35 protein
Arabidopsis thaliana At2g03890/T18C20.9
protein
Homo sapiens protein (Homo sapiens cDNA
FLJ12547 fis, clone NT2RM4000634)
Arabidopsis thaliana protein (Arabidopsis
thaliana genomic DNA, chromosome 5, P1
clone MNB8)
KS050153T0 KS05 Capsicum annuum cDNA
KS06003243 KS06 Capsicum annuum cDNA
4C9 Capsicum chinense subtraction library
Capsicum chinense cDNA
65
EPMS480
BM062655
U204870
RNA recognition motif containing protein

U198888
Expressed protein G86340 protein F2D10.35

U196545

EST homologs
Tomato
Potato{
BI926607*
BM406842*
BI203623*
BM113158*
AW616965*
BI175800*
AW039917
BE923988
CO907720
U197913
U205114
DN980489
Ethylene-responsive DEAD box RNA helicase

Expressed protein At1g72650/F28P22_16
AI491087
BE432717*
BI178058
No.
49

Unigene
No.
BLAST annotation
U196623
31 kDa ribonucleoprotein, chloroplast precursor
U199700
U196493
Glyceraldehyde-3-phosphate dehydrogenase,
cytosolic (Arabidopsis thaliana)
U200239
RNA recognition motif (RRM)-containing protein (Arabidopsis thaliana)
U198459
Thioredoxin family protein T00482
U202943
Nagy et al.
Unigene
No.
BLAST annotation
U198650
Glutamyl-tRNA amidotransferase (Arabidopsis
thaliana)
U198040
No.
66
Code
EPMS490
Accession
No.
BM066956

67
68
69
70
71
72
73
EPMS492
EPMS497
EPMS501
EPMS507
EPMS514
EPMS538
EPMS539
CA517063
CA522759
CA523558
CB164833
CB185070
BM068084
BM068544

KS050013T0 KS05 Capsicum annuum cDNA
KS04239T70 KS04 Capsicum chinense cDNA
74
75
76
77
EPMS540
EPMS542
EPMS543
EPMS546
CA514054
CA514614
CA515275
CA516696

U197649
U198029
U201138
U196717
U203478
78
EPMS547
CA516837
U197406
79
EPMS549
CA517430
U196997
80
81
82
83
EPMS554
EPMS556
EPMS557
EPMS558
CA523715
CA525274
CA525515
CA525592

U202664
U198622
U201040
U201943
84
85
EPMS561
EPMS562
BM068460
CA524381

U204797
86
EPMS563
BM061461
U196664
U196969
AAA-ATPase-like protein (Oryza sativa)
U196336
U198885
U199268
bZIP transcription factor (Capsicum chinense)
Serine/threonine protein phosphatase PP2A

catalytic subunit protein
(Nicotiana tabacum)
GAI-like protein 1 (Vitis vinifera)
Putative thioredoxin m2 (Pisum sativum)
Poly(A)-binding protein (Cucumis sativus)
Expressed protein similar to unknown protein
AAA33773.1 (Arabidopsis thaliana)
Phototropic response protein family
Putative aleurone ribonuclease (Oryza sativa)
mRNA binding protein precursor (Solanum

lycopersicon)
EST homologs
Tomato
AW218125
Potato{
CK248403
BG590247*
CK263920
AW995353
AI775385
BM407743
Bactericidal permeability-increasing protein

precursor related (Arabidopsis thaliana)
Arabinogalactan-protein precursor (Nicotiana
alata)
U199127
Note: Identifiers of pepper unigene builds and their BLAST annotations were determined using the Web-based utility of the SOL Genomics Network (http://www.sgn.cornell.edu).
*Microsatellite motif in conserved position.
{
Shorter or imperfect microsatellite motif in conserved position.
685
2007 NRC Canada
686
Fig. 3. Alignments of orthologous microsatellite loci. (A) EPMS375: CA516199 (Capsicum annuum), CK275337, CK259504, CK259503,
CK260251, CK260250 (Solanum tuberosum), AI772610, AI896145, BG134388, BI422142 (Solanum lycopersicum). (B) EPMS565:
BM065457 (Capsicum annuum), BQ513455, BM111568 (Solanum tuberosum), BE450047, AW650048, BE450054, AW625915,
AW647883, AW647884, BE450060 (Solanum lycopersicum).
query sequences were blasted against 9 554 clustered pepper unigenes available at the SOL Genomics Network
(http://www.sgn.cornell.edu). The results of the homology
searches are summarized in Table 6, which shows EMBL
accession numbers, descriptions of the pepper source sequences as well as the tomato and potato orthologs, and
annotations of the corresponding pepper unigenes.
Out of 86 polymorphic EPMS markers, 35 (40.6%) highly
conserved (at least 150 bit score, Expect value > e50) tomato and (or) potato counterparts were found. In the cases
of 12 potato and 12 tomato orthologs, the sequences contained homologous microsatellite motifs in conserved positions as well. It is interesting to note that in almost every
case the pepper ortholog contained the longest and the tomato ortholog the shortest microsatellite motif. This might
be an effect of the large genome size of Capsicum spp. in
relation to the other solanaceous species (see Fig. 3 for 2 examples). Cross-species transferability of microsatellite
markers has been demonstrated within several plant families
including Leguminosae (Peakall et al. 1998; Kolliker et al.
2001), Cucurbitaceae (Katzir et al. 1996), Triticaceae (Saghai Maroof et al. 1994; Roder et al. 1995; Thiel et al.
2003), and Solanaceae (Provan et al. 1996; Smulders et al.
1997).
From the highly saturated genetic maps of tomato and potato, important information can be derived for the positional
mapping of pepper genes as well. Earlier comparative mapping studies within the Solanaceae (Tanksley et al. 1988,
1992) indicated high synteny throughout the whole genome
of both tomato and potato, but more radical rearrangements
for the pepper chromosomes. For a precise understanding of
this phenomenon, a high number of transferable polymorphic markers and the improvement and further saturation of
existing pepper genetic maps are necessary. A relatively

large number of conserved ortholog set (COS) markers
(Fulton et al. 2002) are available for comparative mapping
in solanaceous species. However, to date no published results are available concerning genetic mapping of COS
markers in pepper.
Presently there are a limited number of pepper SSR
markers in the public domain, and only a portion of these
markers can be used for genetic mapping experiments. In a
recent study of 76 functional SSR markers, 46 could be
mapped using an interspecific mapping population (Lee et
al. 2004), but these markers identified only 19 distinct loci
because some of the SSR markers were located in clusters.
Based on the data of 6 different inter- and intra-specific pepper maps, an integrated genetic map was recently constructed (Paran et al. 2004) based predominantly on
amplified fragment length polymorphisms, which are difficult to transfer owing to the dominant and multi-locus nature of this marker class.
The transferable SSR markers described in this paper are
potentially useful tools for the comparison and integration of
genetic maps of the solanaceous species.
Acknowledgements
The authors are indebted to Tatyana Areshchenkova for
her contribution to the development of genomic microsatellite markers and to Alain Palloix (INRA Montfavet, France),
Gabor Csillery (Budakert Ltd., Budapest, Hungary), and
Zsolt Sagi and Pal Salamon (Vegetable Crops Research Institute Station, ZKI, Budapest Hungary) for providing pepper inbred lines and wild species. This work was supported
by the Hungarian Ministry of Education NKFP program
#
2007 NRC Canada
Nagy et al.
Gold of Hungaria, grant No. 4/006. I.N. acknowledges the

support of the Deutsche Forschungsgemeinschaft and the
Hungarian TeT Foundation.
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2007 NRC Canada

Development, Characterization, and Transferability To Other Solanaceae of Microsatellite Markers in Pepper (Capsicum Annuum L.)

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Development, Characterization, and Transferability To Other Solanaceae of Microsatellite Markers in Pepper (Capsicum Annuum L.)

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Development, characterization, and transferability

author (e-mail: inagy1@gmx.net).

Genome 50: 668688 (2007)

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man 1987). As a result, microsatellite loci often mutate by

Materials and methods

Plasmid clones were sequenced on an ABI PRISM1 377 or

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Genome Vol. 50, 2007

Table 1. Genotypes used for polymorphism investigations.

MK1 (sweet conical

C. baccatum var. pendulum

be functional on this primary test panel (i.e., it produced

Table 2. Frequencies of microsatellite motifs in

Note: n.a., not applicable; n.d., not determined.

2,7-dimethoxyfluorescein) were synthesized for selected

where Pi is the frequency of the ith allele and k is the total

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Primer pairs tested

Results and discussion

348 dinucleotide, 519 trinucleotide, 15 tetranucleotide, and

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Forward primer / reverse primer (5?3)

Capsicum spp. (33 genotypes)

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Capsicum annuum (14 genotypes)

(TC)6. . .(TC)5. . .(TC)6

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Forward primer / reverse primer (5?3)

Capsicum spp. (33 genotypes)

Capsicum annuum (14 genotypes)

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(TC)6. . .(CT)4. . .(TC)7

Forward primer / reverse primer (5?3)

Capsicum spp. (33 genotypes)

Genome Vol. 50, 2007

Capsicum annuum (14 genotypes)

Forward primer / reverse primer (5?3)

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Forward primer / reverse primer (5?3)

Genome Vol. 50, 2007

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Forward primer / reverse primer (5?3)

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Forward primer / reverse primer (5?3)

Genome Vol. 50, 2007

Forward primer / reverse primer (5?3)

dancy within this repeat class was calculated to be 1.37.

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Genome Vol. 50, 2007

Genetic diversity in Capsicum revealed by microsatellite

one was a more morphologically heterogeneous subgroup

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lum) were located on 2 different forks of the same branch

num pimpinellifolium, and Solanum tuberosum. In all, 44%

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Table 6. Homology relationships of the informative EPMS markers.

Related pepper unigenes

KS01023E09 KS01 Capsicum annuum cDNA

KS01027C08 KS01 Capsicum annuum cDNA