Development, characterization, and transferabilityto other
Solanaceae of microsatellite markers inpepper (Capsicum annuum
L.)Istva n Nagy, Aniko Sta gel, Zsuzsanna Sasva ri, Marion Ro der,
and Martin GanalAbstract: A novel set of informative
microsatellitemarkers for pepper (Capsicum annuum L.) is provided.
Screening ofapproximately 168 000 genomic clones and 23 174 public
database entries resulted in a total of 411
microsatellite-containingsequencesthat couldbeusedfor primer
designandfunctional testing. Aset of 154microsatellitemarkers
originatedfromshort-insert genomiclibraries and257markers
originatedfromdatabasesequences. Of thosemarkers,
147(61fromgenomiclibraries and86fromdatabasesequences)
showedspecificandscoreableamplificationproductsandde-tectedpolymorphismsbetweenat
least 2of the33linesof atest panel consistingof
cultivatedandwildCapsicumgenotypes. Theseinformativemarkers
weresubsequentlysurveyedfor allelicvariationandinformationcontent.
Theusefulnessof thenewmarkers for
diversityandtaxonomicstudieswasdemonstratedbytheconstructionof
consistentphylogenetictreesbasedonthemicrosatellitepolymorphisms.
Conservationof asubset of microsatelliteloci inpep-per, tomato,
andpotatowasprovenbycross-speciesamplificationandsequencecomparisons.
For several informativepepper microsatellitemarkers,
homologousexpressedsequencetag(EST) counterparts
couldbeidentifiedinthesere-latedspeciesthat
alsocarrymicrosatellitemotifs.
Suchorthologscanpotentiallybeusedasreferencemarkers
andcommonanchoringpointsonthegeneticmapsof different solanaceous
species.Key words: pepper, Capsicum spp., microsatellite(SSR)
markers, diversity studies, polymorphism, cross-species
transfer-ability.Resume:
Unenouvellecollectiondemicrosatellitesinformatifschezlepoivron(CapsicumannuumL.)est
decrite.Lecriblagedenviron168000clonesgenomiqueset
23174entreesdanslesbanquesdedonneespubliquesapermisdidentifier
411sequencescontenant desmicrosatellites, deconcevoir desamorceset
detester leurfonctionnement.Unjeude154marqueursmicrosatellitesaete
derive debanquesgenomiquesa` insertsdepetitetaillealorsque257ont
ete obtenusdelabanquededonnees. Decenombre,
147marqueurs(61desbanquesgenomiqueset 86desban-quesdedonnees)ont
produit desampliconsspecifiqueset lisibleset ont permisdedetecter
dupolymorphismeentreaumoinsdeuxdunpanel de33ligneescomprenant
desgenotypescultiveset sauvagesdugenreCapsicum. Lutilite
decesnouveauxmarqueurspourdesetudesdediversite et detaxonomieaete
demontreeenproduisant
desarbresphylo-genetiquesconcordantsbasessurlepolymorphismedesmicrosatellites.
Laconservationdunsous-ensembledeslocusmicrosatellitechezlepoivron,
latomateet lapommedeterreaete
montreeparamplificationinterspecifiqueet lacomparaisondessequences.
Pourplusieursmicrosatellitesinformatifsdupoivron,
desetiquettesdesequenceexprimee(EST)homologuescomprenant
desmicrosatellitesont ete identifieeschezcesespe`cesapparentees.
Detelsortholo-guespeuvent potentiellement
servirdemarqueursdereferenceet
depointsdancragesurlescartesgenetiquesdesdif-ferentesespe`cesdesolanacees.Mots-cles
: poivron, Capsicum spp., marqueurs microsatellites( SSR ), etudes
de la diversite, polymorphisme,
transpor-tabiliteinterspecifique.[Traduit par la
Redaction]IntroductionMicrosatellites or simple sequence repeats
(SSRs) areDNAsequences consistingof tandemlyrepeatedarrays ofshort
(16nucleotides) motifsthat frequentlyexhibit
poly-morphismbetweencloselyrelatedgenotypes. This polymor-phismis
probably due to slipped-strand mispairing,
andsimpletandemrepeatsmaybepredisposedtofurtherlengthchangesbyunequalcrossingoverand(or)subsequentrepli-cation,
repair, or recombinationerrors (LevinsonandGut-Received 4 December
2006. Accepted 12 June 2007. Published on the NRC Research Press
Web site at genome.nrc.ca on 3 August2007.Corresponding Editor: P.
Gustafson.I. Nagy,1A. Stagel, and Z. Sasvari. Agricultural
Biotechnology Center, Szent-Gyorgyi Albert u. 4, H-2100 Godollo,
Hungary.M. Roder and M. Ganal.2Institut fur Pflanzengenetikund
Kulturpflanzenforschung, Corrensstrae 3, D-06466 Gatersleben,
Germany.1Corresponding author (e-mail:[email protected]).2Present
address: TraitGeneticsGmbH, Am Schwabeplan 1b, D-06466 Gatersleben,
Germany.668Genome 50: 668688 (2007) doi:10.1139/G07-047 # 2007 NRC
Canadaman1987). Asaresult, microsatelliteloci
oftenmutatebyinsertionsordeletionsofoneormorerepeat elements,
andthemutationratesgenerallyincreasewith anincreaseinthelength of
the repeats (Wierdl et al. 1997; Xu et al. 2000).
Aslocus-specificcodominant markersthat
canbeanalyzedbyhigh-throughput PCR-based techniques,
microsatellites areidealtools for differenttypes of
fingerprintingand genotypeidentificationtasks as wellasfor
constructionof frameworkmaps and for marker-assisted breeding
(Rafalski et al.1996). The benefits of using informative
microsatellitemarkers for saturation and integration of existing
geneticmapshavebeendemonstratedinanumberofplant
speciessuchaswheat(Roderetal. 1995, 1998;Guptaetal. 2002),rice
(Temnykh et al. 2000), barley (Pillen et al. 2000; Ramsayet al.
2000; Li et al. 2003), maize(Sharopovaet al. 2002),Cucumis spp.
(Gonzalo et al. 2005), and tomato (BrounandTanksley1996;
AreshchenkovaandGanal 1999, 2002;Fraryet al. 2005).Pepper
(CapsicumannuumL.) isanimportant vegetablecrop worldwide. Besides
having relatively large genomes(3753to4763Mbindifferent
Capsicumspecies, Bennettand Leitch2005), cultivatedpepper genotypes
also exhibit alow level of molecular polymorphism; the lack of
hypervari-ablemolecular markersinpepper hamperstheconstructionof
saturated genetic maps. Although activities directed
towardmicrosatellite marker development andmicrosatellite
map-pinginpepper werereportedrecently(Huanget al. 2000;Lee et al.
2004; Ogundiwin et al. 2005), and
proprietarymicrosatellitemarkershavebeenmadeavailablefor diver-sity
studies and genetic mapping (Tamet al. 2005; BenChaim2005),
thenumberofpresentlyavailablepepper mi-crosatellite markers is far
fromthe number that wouldbenecessaryfor
high-densitymappingandefficient genetar-geting.In this paper we
report the development and primary char-acterizationofanovel set
ofpepper microsatellitemarkersobtainedfrom genomiclibrariesand
publicly availabledata-basesequencesaswell
asdataconcerningthetransferabil-ityofthesemarkerstoothersolanaceousspecies.Materials
and methodsLibrary
screeningsGenomicDNAfromthepeppercultivarsC.annuumFe-herozon andC.
annuum Blondywas digestedwith there-strictionenzyme MboI or Sau3AI.
Size-selectedfragmentsbetween400and1000bpwereligatedintotheBamHIsiteof
the ZAPExpress1vector (Stratagene). Packaging intophages, plating,
hybridization, and in vivo excision wereperformed according to
Roder et al. (1995, 1998).The construction and management of
plasmid librariesenriched in single-copy sequences and the
preparation
ofhigh-densityhybridizationfiltersusingtheBioPick/BioGridroboticsystem(BioRoboticsLtd.,
UK) werecarriedout asdescribed by Pestsova et al. (2000). Filters
were subse-quently hybridized with32P-labeled synthetic
oligonucleo-tide 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
fortrinucleotide motifs; and (AACT)4, (ACAT)4, (GATA)4,(GACA)3,
(GGCC)3, and (GGTT)4 for tetranucleotide motifs.Plasmid clones were
sequenced on an ABI PRISM1377 or3100 automatic sequencer (Applied
Biosystems) usingstandard sequencing procedures.Database
searchesNucleotide sequences belonging tothe genus Capsicumwere
retrievedfrom the EMBL databaseusing the SequenceRetrieval System
(SRS) to build a stand-alone nucleotide da-tabase that could be
searched using the utilities of theBLAST2 package (Altschul et al.
1997). All further databasemanagementand DNA
sequencemanipulationswere carriedout onlocal PCs under the
LinuxoperatingsystemusingstandardUNIX tools and
public-domainbioinformaticssoft-ware. Searches toidentifyentries
containingshort
tandemrepeatswereperformedbythefuzznucnucleicacidpatternsearchprogramoftheEMBOSSsoftwarepackage(Riceetal.
2000).Data
processingMicrosatellite-containingcloneswereinspectedindividu-ally
with respect to sequencequality and suitabilityfor
PCRamplification. Redundant clones werefilteredout bylocalBLAST
analyses using the individual clones as queriesagainst the whole
set of repeat-containing sequences.
Primerpairsflankingthemicrosatellitemotifsweredesignedusingthe
Primer3 program (release 0.9, Rozen and Skaletsky 2000).Plant
materialTomeasure the level of microsatellite polymorphism, aset of
33 pepper genotypes was used, consisting of cultivatedvarieties,
inbredlines, andwildCapsicumspecies.For testing the cross-species
transferability of the markers,all developedmicrosatellitemarkers
were tested for amplifi-cation in the followingspecies of the
family
Solanaceae:Solanumpimpinellifolium(formerlyLycopersiconpimpinel-lifolium),
inbred line WaWa700; Solanum lycopersicum (for-merly Lycopersicon
esculentum), inbred line 4889; andSolanum tuberosum Desiree
(Table1).Analysis of microsatellite polymorphismsDNA was isolated
from leaves of greenhouse plants usingstandard procedures
(Dellaporta et al. 1983). PCR amplifica-tionswerecarriedout
in25mLvolumeswith1050ngofgenomic DNAas template. Reaction mixtures
contained10mmol/LTrisHCl(pH8.3), 50mmol/LKCl, 2.5mmol/L MgCl2, 0.5
U Taq polymerase, 200 nmol/L of the unlabeledreverse primers, 160
nmol/L of the fluorescently labeledforwardprimers,
and0.2mmol/Lofeachdeoxynucleotide.Aninitial denaturationstepat
948Cfor 3minwas typi-callyfollowedby35cyclesof 928Cfor 1 min,60 8C
for1min, and728Cfor1min.Thereactions wereterminatedbyafinal
extensionstepat 728Cfor7min.For the first functional tests,
unlabeledforwardprimerswere synthesized, andDNAof the parental
andF1geno-typesofaninterspecific(C.frutescensTabasco
C.ann-uumP4)andintraspecific(C. annuuumCM334 GF109)hybrid
combination was used as template. Amplificationproducts were run on
agarose gels and (or) on ALFex-pressTMII sequencers (GEHealthcare
Life Sciences) withthe incorporation of Cy5-labeled dCTP (GE
HealthcareLifeSciences). Incasesinwhichanewmarker provedtoNagy et
al. 669# 2007 NRC Canadabe functional onthis primary test panel
(i.e., it producedwell-defined fragments and showed at least 1
polymor-phismbetweenthe4parental genotypes), fluorescentlyla-beled
forward primers were synthesized to enable furtheranalyses on
automatic sequencers. Fragment analysis ofPCRproducts labeledbythe
cyanine dye
Cy5usingAL-FexpressTMIIsequencerswascarriedoutasdescribedelse-where(Li
et al. 2003).To facilitate multiplexed semi-automated analysis
oflargernumbersofsamplesonanAppliedBiosystems3100capillary
sequencer, forward primers labeled with FAM(5-carboxyfluorescein)
or JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein) were
synthesized for selectedpolymorphic markers. Amplification products
were ana-lysedafter simultaneouslyrunningFAM-
andJOE-labeledPCRproducts and ROX(5-carboxy-X-rhodamine)
labeledinternal size standards on 36 cmcapillaries with POP4polymer
at 1500Vusinga15s injectiontime. Fragmentanalysis data
fromcapillary runs were collected by thebuilt-in data collection
software andpre-processed bytheGeneScan software (version 3.7,
Applied Biosystems).GeneScandatawereimported, convertedtopseudogel
im-ages, and further analyzed by the Genographer
program(Benham2001).Polymorphisminformation content(PIC) valueswere
cal-culatedaccordingto the following equation(Anderson et al.1992;
Hedrick 1985):PIC 1
Xki1Pi2wherePiisthefrequencyoftheithalleleandkisthetotalnumber of
different alleles at the locus. For each pepperSSRmarker,
2different PICindices werecalculated: oneTable 1. Genotypes used
for polymorphism investigations.Species No. GenotypeInbred
linesCapsicum annuum 1 MK1 (sweet conicalwax)2 MK2 (yellow blocky)3
MK3 (tomato shape)4 MK4 (white apple)5 MK5 (Lamuyo type)6 MK6 (red
paprika)7 MK7 (dark greenblocky)8 MK8 (long pale green)9 Yolo
Wonder (green tored blocky)10 Perennial (chile)11 P4 (red
paprika)12 GF109 (red paprika)13 CM334 (chile)14 MN1 (purple leaf
orna-mental)Wild Capsicum genotypesC. frutescens withC. chinense
introgression15 TabascoC. chinense 16 chi117 chi718 chi2119 chi25C.
eximium 20 exi1-104321 exi1-1052C. pubescens 22 pub223 pub324
pub5C. baccatum var. pendulum 25 pen126 pen6C. praetermissum 27
pra1C. chacoense 28 cha629 cha9C. baccatum var. baccatum 30 bac1031
bac20C. frutescens 32 fru3933 fru48Solanum genotypesSolanum
pimpinellifolium 34 WaWa700Solanum lycopersicum 35 4889Solanum
tuberosum 36 DesireeTable 2. Frequencies of microsatellitemotifs
inpepper (Capsicum spp.) sequences in the EMBLdatabase and in
genomic libraries.Frequency*RepeatDatabase(23 174 entries)Genomic
libraries(55 296 clones)A 181 n.a.C 8 n.a.AC 17 12AG 104 11AT 29
10{AAC 21 0.5AAG 60 0.5AAT 20 7ACC 37 2ACG 2 0ACT 7 0.5AGC 24
0.3AGG 12 0.3ATC 27 0.3CCG 15 n.d.AAAC 0 0.3AAAG 2 n.d.AAAT 3
n.d.AACT 0.4 0.6AAGG 0.4 n.d.ACAT 0 0.3ACAG 0 0.3ATAG 0 0.3AATT 0.4
n.d.AGGG 0.4 n.d.AAAAG 0.4 n.d.ACTAT 0.4 n.d.Total 571.4 46.2Note:
n.a., not applicable; n.d., not determined.*Occurrence per 10 000
clones or entries.{Not screened but found randomly.670 Genome Vol.
50, 2007# 2007 NRC Canada(PICa)fortheinformationcontent inC.
annuum(14geno-types)andone(PICc)forall
Capsicumgenotypesfromthesecondarytest panel
(33genotypesbelongingto8species,including the 14 lines of C.
annuum).Cluster analysisThepresenceor absenceof eachSSR allelewas
coded as1 or 0, respectively. The resulting binary matrix was
directlyused to construct unrooted phylogenetic trees with the
DOL-LOPprogram.
Pheneticanalysiswasperformedaftercalcu-latingpairwisegeneticsimilarityindices(Nei
andLi 1979)fromthebinarymatrix, usingtheUPGMAmethodoptionof the
NEIGHBORprogram. Consensus trees wererecon-structed bythe
CONSENSEprogram; graphical renderingof the dendrograms was
processed by the DRAWTREE pro-gram (PHYLIP package,version 3.6,
Felsenstein 2005).Results and discussionGenomic
librariesApproximately60 000phageclonesand108 000plasmidclones were
used for the screening of microsatellite-containingsequences.
Basedonanaverageinsert sizeof550bp, 80%of plasmids containing
inserts, and 10%redundancy, thetotal length of the screened genomic
sequences was estimatedto be 66 000 kb. The screenings revealed 512
positiveclones,whichcorrespondedtoonepositivecloneforevery129kb.However,
formost ofthescreeningsonly(GA)nand(GT)ndinucleotide repeat probes
were used for the plaque andcolonyhybridizations, andanumber of
thepositiveclonesprovedtobefalsepositivesaftersequencing.
Todeterminemore representative frequency estimates, 3 high-density
filterscontaining 55 296 colonies were sequentially probed withdi-,
tri-, and tetra-nucleotide repeats. All positive cloneswere
isolated and sequenced and microsatellite-containingclones fromthe
test filters were consideredfor
frequencycalculations.Microsatellitemotif frequenciesAt
thetimeofthesurvey, theEMBLsequencedatabase(release76)contained23
174entriesbelongingtothegenusCapsicum.Themajorityoftheseentries(about98%)repre-sentedshort
partial cDNAsequences(conventionallycalledexpressedsequence tags or
ESTs). The total lengthof allCapsicum sequences reached 10 828 kb.
Search criteriawere set to identify microsatellite motifs of at
least 16mononucleotide, 8dinucleotide, 5trinucleotide,
5tetranu-cleotide, or 5pentanucleotiderepeats, allowingfor
onlyasingle mismatch. The searches resultedina total of
1322microsatellites, of which 438 represented
mononucleotide,348dinucleotide, 519trinucleotide,
15tetranucleotide, and2pentanucleotide repeats. Thirty-nine
sequences containedmorethanonemicrosatelliteand37SSRs
occurredincom-pound form
(excludingmononucleotidestretches).A/Tmononucleotiderepeats
werethemost frequent mi-crosatellitemotifsinthedatabasesequences.
Toavoidtar-getingpoly(A)tailsofcDNAsequences, searchcriteriaforA/T
mononucleotiderepeats were set so that only sequencesthat contained
A/T repeats at least 20 nucleotides away fromeitherthe5 orthe3
endwereselected. Withsuchcriteria,in almost 2% of the database
sequences, A or T repeats longerthan 16 bp were found. The
frequency of C/Gmononu-cleotide repeats was much lower (less than
0.01%). Screeningof genomic libraries for mononucleotide repeats
was notundertaken. Thesamewastrueforthe(AT)nhybridizationprobe
owing to its self-complementarity. However, (AT)nrepeat
motifscanbefoundrelativelyfrequentlyat
randominthesequencedgenomicclones, inthemajorityof casesas part of
compoundrepeats. Thefrequencyof compoundSSRs is strikingly high in
pepper genomic clones and,most typically, (AC)n or (AG)n
microsatellites are associatedwith(AT)nrepeats.Trinucleotide
repeats are the most abundant class of repeatmotifsinall
cDNA-dominatedsequencedatabases(Chenetal. 2006; Choet al. 2000;
Thiel et al. 2003). Inour study,the frequency of all specific
trinucleotide repeats was39.2%,
whilethefrequencyofmononucleotiderepeatswas33.1%and that of
dinucleotide repeats was 26.3%, fromthetotal
of1322identifiedmicrosatellites.All 10possible types of
trinucleotide repeat motifs oc-curredindatabasesequencesof pepper.
Themost frequentrepeat types inpepper wereAAG, ACC, andATC,
whileACG and ACT repeats were relatively rare. Similar
frequencypatternswerefoundintheglobal plant sequencedatabases(Katti
et al. 2001; Toth et al. 2000), while CCGrepeatsseemed to dominate
monocot ESTsequences (Cho et al.2000; Thiel et al. 2003). The
occurrence of
trinucleotiderepeatswasgenerallylowingenomiclibraries.
Microsatel-lites with5or morerepeat unitsof tetra-
andpenta-mericrepeatswerefoundat
verylowfrequencyinbothtypesofsequence pools. The frequencies of
different repeat typesfoundingenomic libraries andindatabase
sequences aregiveninTable2.Toget
someinformationonpossiblefrequencyoveresti-mations,
aredundancyanalysisfordatabasesequenceswasperformedforthemost
prominent dinucleotiderepeat type,(AG)n. Among240identified
sequences containing(AG)nrepeats, 27 (11%) were found to be present
redundantly,withanaverageoccurrenceof
3.4times.Theoverallredun-Table 3. Functionality of the newly
developed microsatellite markers on a test panel consistingof
33Capsicumgenotypes.Genomic libraries Database sequencesNo. % No.
%Primer pairs tested 154 257No amplification 29 18.8 42 16.3Weak or
unspecific products, not scoreable 40 26.0 109 42.4Monomorphic 24
15.6 20 7.8Functional markers 61 39.6 86 33.5Nagy et al. 671# 2007
NRC CanadaTable 4. Allelic variation and information content of the
informative GPMS markers.Capsicum annuum (14 genotypes) Capsicum
spp. (33 genotypes)No. Code Repeat Forward primer / reverse primer
(5?3)Allele sizerange (bp) No. of alleles PICa No. of alleles PICc1
GPMS1 (AC)18 CCCTAATGCTTGACGTGG/ 121163 4 0.49 11
0.83GGTTAAGGGGGTTGGGG2 GPMS3 (CA)17(TA)21 ACTTGACAGTCGTGTATCTGCA/
262282 1 0 6 0.73GGACTCCACCAGCTCAGTTC3 GPMS4 (GT)8GCG(TG)5(TA)7
TTGATTTTTAAAGCAAAGTCGG/ 196216 1 0 7 0.71ACAAGGAAATTCTTGCAGGG4
GPMS6 (AC)20imp(AT)5 CAGAGCACTTGACATGCCTT/ 122172 4 0.68 12
0.89GATCTTTATAGTAGCTCATCAATA5 GPMS8
(AT)9(GT)22TGATGATAAGGCCATGATAAAATG/ 159229 6 0.77 14
0.87CCAGATTCTTTAGCAAGGTTTACC6 GPMS29
(GT)15GGT7(GTT)2CAGGCAATACGGAGCATC/ 238255 4 0.60 7
0.83TGTGTTGCTTCTTGGACGAC7 GPMS37
(TC)18(TA)12ATTTGTATATTATTTCTTGGCCTTG/ 176182 3 0.60 4
0.67TGAACTACCCAATTCCAGCC8 GPMS93
(TA)14imp(GA)27impATCCTTGGCGTATTTTGCAC/ 202268 3 0.44 12
0.86TTCACTTTGCACACAGGCTT9 GPMS100 T5(GT)12TCCATACGGTTGGAGGAGAG/
141169 4 0.57 7 0.83ACTATGCTCTGCTGTGCCCT10 GPMS101
(TC)16impCCTATCACCCTCTTTGAGCC/ 176211 2 0.46 6
0.67TAAAGACCAGCCCTGGATGA11 GPMS103 (CT)11TGGCAGTTGCAATATCAATCTC/
134149 1 0 6 0.70AAACCTTGTCACGCATCCAT12 GPMS104
(AT)6(GT)11GGGTCATGGGATTTCTTTTC/ 96123 2 0.13 6
0.72ATTCAAACCATCCTTCAAGTTC13 GPMS107
(AT)7(GT)12AACTAATTCTTGTCTGAGCA/ 177182 2 0.13 4
0.62CATTTAACTTGATTTGATTG14 GPMS109 (AC)14(AT)7ATCTATGCATGCCATCACCG/
165183 2 0.13 2 0.48TGGGCTAAAGGCCATGTTAC15 GPMS111 (AT)7(GT)12
TCAGAAGATGCCCATGTGTT/ 120132 1 0 2 0.17TACTGGCACACGAAGCAATC16
GPMS112 (AT)8(GT)19 TCCCTCAGCAGCAACAATTT/ 203280 5 0.76 10
0.86GTCGGGCTCTTTGATTGTGT17 GPMS113 (AT)20(AG)18
GCACAAGTCAATCCAAACGA/ 91172 9 0.86 15 0.90CAAAAAGATGATGATGGATGAGA18
GPMS117 (TA)25(GA)14 GATGTTAGGTCCGTGCTTCG/ 111177 5 0.76 11
0.86AAGCCCCATGGAAGTTATCC19 GPMS119 (GT)11G13 CTGGAATCTGTCAATTGGTTG/
204224 3 0.54 6 0.81TCGTTTCATGATGGAATTGG20 GPMS140
(TA)7imp(CA)7(TA)4 AACACATCGGTGGGTAATCC/ 169199 1 0 6
0.63TGATTCCACCAGGCTTTAGG672GenomeVol.50,2007#2007NRCCanadaTable 4
(continued).Capsicum annuum (14 genotypes) Capsicum spp. (33
genotypes)No. Code Repeat Forward primer / reverse primer
(5?3)Allele sizerange (bp) No. of alleles PICaNo. of alleles PICc21
GPMS141 (AC)7(CA)2(TA)5CATACATACACACGCGCATG/ 100149 1 0 8
0.76TGAAATACGTTGTAATTATTATGGG22 GPMS142
(CA)6(TA)4TTCGACCTCTTGGTATTCCG/ 103125 1 0 5
0.56CACGACACATGGACTCCTTG23 GPMS147 (CT)19 AGGAGGATATTTTGGCAGCC/
184240 1 0 7 0.69AAAAACACCCATGGGAAGTG24 GPMS150 (AT)4(AC)9. .
.(AT)33 TGAGCGCTAACATATGTCGG/ 291299 1 0 4
0.58GGCCCTTCCTCAGATTTCTC25 GPMS151 (TC)13 ATCCTCGAACTTTGGTGTGG/
183191 1 1 5 0.58TGCATGCTCAGCAGGTAGAG26 GPMS153 (AC)10imp(AT)9
TGCTAGCTTCAACTGGTCCC/ 200225 1 0 3 0.26TCAAATGTATGCATGCTTTGC27
GPMS154 (CA)4A(CA)9 AATCTAATTTCTCGCCTGCG/ 165178 2 0.50 5
0.67ATCACATTTTGGTTTTGGGG28 GPMS155 (TG)12impCA(TG)4
GGGCGAATCGAGCTACATAG/ 171173 2 0.13 2 0.06GGGATAGCGACAAAAAGTGC29
GPMS156 (GA)6(CA)6GATATAGAACCACACAAGACTGGG/ 228232 2 0.13 3
0.64TATGGCTCTTGCGGGAATAC30 GPMS157 (TG)7AATGCGCTGAAACCCAATAC/
278305 1 0 8 0.68TTTGAGGAAACACTAAAAAGTGTCC31 GPMS159
(TAA)20AAGAACATGAGGAACTTTAACCATG/ 281317 5 0.61 10
0.82TTCACCCTTCTCCGACTCC32 GPMS161 (AAT)25CGAAATCCAATAAACGAGTGAAG/
184259 5 0.8 12 0.89CCTGTGTGAACAAGTTTTCAGG33 GPMS162
(TA)2(TATG)4(TG)5TAACACACGCATGGTGAACC/ 305325 2 0.13 6
0.64TCGGTTATCTTATACGGCCTG34 GPMS163 (AAC)5CCACCGCTATCACTACCACC/
280291 1 0 4 0.57CCTCCGCAGTAGTGGTATGG35 GPMS164
(ACC)5AATGAAATCAATCGGGCTTG/ 230259 1 0 5 0.47ACCTCGCACCAATTCTTTTG36
GPMS165 (TAA)35imp. . .(TA)26TGAACAATAATAATTGACAGGACAG/ 242317 5
0.61 13 0.87AGCCTCGCAGTTTGTTCTTAC37 GPMS166
(ACC)7AAAACCGACACACCAAAAGC/ 193247 3 0.36 10
0.82CCCTAGTTTCCGTTGCAGAG38 GPMS169
(ATT)5T(TTA)7TCGAACAAATGGGTCATGTG/ 176220 3 0.52 8
0.78GATGAGGGTCCTGTGCTACC39 GPMS171 (TC)6. . .(TC)5. .
.(TC)6TCCACCACAATATTTCGAAGG/ 288346 2 0.13 7
0.72TGGCTGTCCAACACTGTGAG40 GPMS176 (TAT)13 CCGCATTCTTGATTGAAACG/
327345 2 0.13 7 0.70TGGGCAGTATATTGTGCGTG41 GPMS178 (ATT)19
GATTTTTGACATGTCACATTCATG/ 230261 5 0.78 14
0.89Nagyetal.673#2007NRCCanadaTable 4 (concluded).Capsicum annuum
(14 genotypes) Capsicum spp. (33 genotypes)No. Code Repeat Forward
primer / reverse primer (5?3)Allele sizerange (bp) No. of alleles
PICaNo. of alleles PICcAACGTTGAAAAATAAAGTAAGCAAG42 GPMS181 (ATT)5.
. .(ATT)11GAAAGTGGAGAATTTAAAACCTTG/ 166214 2 0.13 8
0.64TTCATTATCTCACCGTTACTGAC43 GPMS183
(TC)9(TA)7GAGCTTCATAGATGATATGCAAGAG/ 195227 1 0 5
0.70TCCCAAGCTAACCCATTTACTG44 GPMS185 T12(GT)7
TCTCCCTGTAATTAGCAGAGCAG/ 238242 2 0.25 5
0.71AAAAATCCCTAAATGAGCAAACC45 GPMS186 (TTA)14 GCGGTAAGGTCTGCGTACAC/
161180 2 0 5 0.52TTTGAAATAAGTTGGGCATAAATG46 GPMS187 (CA)6C4A36
TTTAGAATCCTCACCACGGG/ 219246 1 0 8 0.71TCAATGCACAAACTTTAATTTGC47
GPMS188 (GGT)5(TGG)2 TGCCGTGGTATTATTGGATG/ 121201 1 0 6
0.70ATACCGACACACCAACCACC48 GPMS189 (AT)7imp(AC)7(AT)5
GCTGCATCAAATCCCTAAGC/ 290321 1 0 9 0.68TGATTCCACCAGGCTTTAGG49
GPMS191 (GA)8 AGGTCAGCGACGGCAAC/ 261286 1 0 5
0.54ATTTTAGGAGCCGACCTTCC50 GPMS193 (CTT)8. .
.(CTT)10CGTACAATATCCATCCTTGTGC/ 313335 1 0 5
0.65AAAAAGGAGAAAGATAAGGAAAAGG51 GPMS194
(TA)17(GA)12AGGTGGCAGTTGAGGCTAAG/ 216270 5 0.62 10
0.81GTTCTAGGTCTTTGCCCTGG52 GPMS195 C11(CA)6TGAAAACCAACCACTTGTGG/
231321 1 0 10 0.77TTGGAACTTACCGGAGTTGG53 GPMS196
(TG)7(TA)6AAAGGATGCAACACGAGGAC/ 159208 2 0.13 8
0.72TTCGCCTCACTGTGTTCTTG54 GPMS197
(GA)3(TAT)16GCAGAGAAAATAAAATTCTCGG/ 272344 6 0.81 10
0.82CAATGGAAATTTCATCGACG55 GPMS198 T17(GT)9AGCTTTAGACAGTGTCTGCGTG/
252279 2 0.13 8 0.77TGATGATAAATTGCCTTCCG56 GPMS199
(TA)16(GT)10(GA)16TTGAAGTTGTGATTTTGCATG/ 279371 1 0 12
0.74TTTCCGTAACAAATTTTCAAACC57 GPMS200 (TC)6. . .(CT)4. .
.(TC)7CTGCGACTTAACGCTCACTG/ 233243 1 0 3 0.44AATTAAACCATCGGGAACCC58
GPMS201 (AC)4AT(AC)3ATCAACCCAGCAACACCAAC/ 247250 1 0 2
0.17CTCCAGCAACAACAACTTCG59 GPMS202 (AT)10. .
.(AT)4(TG)9AAAGCTTTCAATAGTTTGGGGAC/ 252305 3 0.56 10
0.87AGGGATAACGTGCAGAGCAC60 GPMS203 (CAC)6CACCAACACATCTTTTTCAACC/
195210 1 0 5 0.68ATAATAGTGGTTGCGGCGAC61 GPMS205 (AC)11(AT)5
TCTCACTCCAAATTGGGGAG/ 313404 2 0.13 10
0.78TGAACCAATATATTTTATACAAACCMean 2.41 0.27 7.28
0.68674GenomeVol.50,2007#2007NRCCanadaTable 5. Allelic variation
and information content of the informative EPMS markers.Capsicum
annuum(14 genotypes)Capsicum spp.(33 genotypes)No. Code Repeat
Forward primer / reverse primer (5?3)Allele sizerange (bp) No. of
alleles PICaNo. of alleles PICc1 EPMS303
(TA)25AAAACTCCAAACTACCCCTGG/ 291330 8 0.85 9
0.84TTAAGCGTAGCGCTTGTGTG2 EPMS305
(CTT)3(CAT)9CGTCTTTCACTTGTCTTTTGTTC/ 94115 3 0.57 8
0.83AGTGGGTTCACTGACTTGGG3 EPMS309 (CTT)6TCATCTTTCTGCAATTCCCC/
224250 2 0.07 7 0.70ACCAGGTTTAGCCCATGTTG4 EPMS310
(CAT)13TGGGAAGAGAAATTGTGAAAGC/ 140172 3 0.44 7
0.81AGGAAACATGGTTCAATGCC5 EPMS316 (AAT)8CACCTCTCTAACCGTCACCC/
155204 2 0.13 4 0.31CAGAGAGCCAGGACAAGACC6 EPMS327
(CGT)7TTTGGAACATTTCTTTGGGG/ 98114 3 0.36 6
0.76ACGTAGCAGTAGGTTTGGCG7 EPMS330 (AT)10TGGGGACCAGATCCAGTTAC/
257268 2 0.13 5 0.67TTTCCGCCTATCAACAATGG8 EPMS331
(CA)10AACCCAATCCCCTTATCCAC/ 92107 5 0.70 8
0.75GCATTAGCAGAAGCCATTTG9 EPMS335 (ACAT)3(AT)17
ATGCAGAGATTGTCGAAGCC/ 286330 4 0.66 10 0.84GCAGAGAAGACTCACCAGTCC10
EPMS340 (AT)13 AAACCTAGTGACTGGCGAGC/ 261288 2 0.24 8
0.52AACGTTTTGCATAACGGAGG11 EPMS342 (CTT)7 CTGGTAGTTGCAAGAGTAGATCG/
323343 4 0.50 7 0.78ATGATCTTTGACGACGAGGG12 EPMS343 (CAT)6
TCTGGGAATTTTGGAACTGC/ 129173 2 0.24 7 0.77TCCAGTTTTGATCATCTCCAAC13
EPMS345 (AAG)7 ATGGACGTTGATGAGCCTTC/ 154163 1 0 4
0.50CGGCCTCCTGTTCTAATTTC14 EPMS349 (GAT)6 TGTAACCAAACTCGTGCTGG/
223235 2 0.07 5 0.62ATCAACCAATCAGCCTCGTC15 EPMS350
(CAT)2(CAA)7AGTGTGGCTACGACAGCATG/ 96106 3 0.3 8
0.75TTGTTTCCCTTACCTGCCAC16 EPMS353 (CTG)6GTATGCTGCAACCATCGTTG/
299317 2 0.13 7 0.70ATTGGTTTGGGAGACACAGC17 EPMS366
(TA)8TTCGTTTTGTTCAGCTGAGAAG/ 171196 1 0 7
0.73AACGATGAATTTGACGTCCC18 EPMS369 (CAT)2. .
.(CAT)6TAATCGAGCGGTAGATTCGG/ 347356 2 0.50 4
0.71TAAGTGGAGGTGCCCTTCTG19 EPMS372 (TA)8TCCACCCAGAAAAAGACTCG/
321325 2 0.24 3 0.60TGGACGGAACAAGAAATTTTG20 EPMS373
(CAT)6TCTCCAATTTCCATTCGGAG/ 177190 1 0 4
0.60Nagyetal.675#2007NRCCanadaTable 5 (continued).Capsicum
annuum(14 genotypes)Capsicum spp.(33 genotypes)No. Code Repeat
Forward primer / reverse primer (5?3)Allele sizerange (bp) No. of
alleles PICa No. of alleles PICcTAATCGCATTTGCGAACTTG21 EPMS374
(CAA)6CAG(CAA)3 TTTTCCCCATGAATAAAGATGG/ 206215 1 0 5
0.69TTCAATTTCACCTTCTGGGG22 EPMS376 (CAA)6ACCCACCTTCATCAACAACC/
235259 5 0.67 9 0.85ATTTGTGGCTTTTCGAAACG23 EPMS377
(AG)11GACAGTCTTTCAAGAACTAGAGAGAG/ 134161 2 0.50 8
0.72TGGAGCAAACACAGCAGAAC24 EPMS378
(CGG)7(AGG)2CGAAGATCACCACCACACTG/ 101110 1 0 4
0.58ACATTTTCACTGCCGGAGAC25 EPMS382 (ACA)9TTTCCCCCATCACATGAAAG/
154166 1 0 5 0.70CCTAAAGCCATGCAAAAACC26 EPMS386
(CA)15ACGCCAAGAAAATCATCTCC/ 122170 5 0.62 11
0.86CCATTGCTGAAGAAAATGGG27 EPMS387
(AAT)2AGT(AAT)6AGGGTTTCCAACTCTTCTTCC/ 162199 2 0.5 10
0.80CTAACCCCACCAAGCAAAAC28 EPMS390 (ATT)8GCTCCTCATTAGTAGCCCCC/
113131 2 0.41 6 0.69TCTTCATCACACCTCTGTGGAC29 EPMS391
(AT)9TTTCTTCTCTGGCCCTTTTG/ 177187 2 0.5 5
0.79ACGCCTATTGCGAATTTCAG30 EPMS395 (CCG)6TGTTGGAGCACCATACTCAAAC/
116131 2 0.24 7 0.46CATCTCCCGACTGAAACTCC31 EPMS396
(CAT)6TCATCGTCTCTTCAACTCTTCTTG/ 223231 3 0.57 4
0.67CCCAATTCGACCTGTTATGG32 EPMS397 (CA)20GCACCCTCCCAATACAAATC/
102117 3 0.54 8 0.76GATCACGGAGAAAGCAAAGG33 EPMS399 (AAT)8
GTTCTTCTCGCCGACAAGTC/ 144175 4 0.37 8 0.81TTCAAAAATCATGAGCAGCG34
EPMS402 (CCT)3(CTT)6(CCT) GCCTTCTTTTTCATCTTTCCC/ 197216 3 0.47 7
0.74CTGGCAACCCAAGTCTTAGC35 EPMS404 (CTT)12 TCTCTCTCTACATCTCTCCGTTG/
215245 5 0.47 9 0.80TGTCGTTCGTCGACGTACTC36 EPMS409 (CAT)7
ATACTAAGGCGTTTGGTCGG/ 162180 3 0.26 8 0.72ACAATTGGGGATGCAGAAAG37
EPMS410 (CT)14(CA)16 imp GGAAACTAAACACACTTTCTCTCTC/ 149187 2 0.54
10 0.85ACTGGACGCCAGTTTGATTC38 EPMS411 (TA)9(GATA)3
AAAGGCAACGATGAGAATGG/ 333373 1 0 8 0.66AGGACACAAGCACACATCATATC39
EPMS412 (AT)9CGGACTTTTCAAAAACACACAC/ 205213 2 0.49 5
0.77ACCGACGGCAATGATCTTAC40 EPMS413 (CCA)9GCCTCATAAAGGTGGCC/ 252268
1 0 3 0.56676GenomeVol.50,2007#2007NRCCanadaTable 5
(continued).Capsicum annuum(14 genotypes)Capsicum spp.(33
genotypes)No. Code Repeat Forward primer / reverse primer
(5?3)Allele sizerange (bp) No. of alleles PICa No. of alleles
PICcAGTTGGCGGTTTAACTGGTG41 EPMS414 (AT)10 TTGGGGACTTCACGTCTCTC/
140186 2 0.07 9 0.77TTGATGATAAATCCTCCCCC42 EPMS415
(CCA)CA(CCA)7GTCGAACAAAATGGGGTTTG/ 212215 1 0 2
0.50GCTGGAGAGTGCTGGTGG43 EPMS416
(CTT)10T(CTT)2GCCAAATGGAGGGTCCTTAG/ 131154 3 0.36 8
0.79CAAAGAAAGATGAAGAAGCAACAG44 EPMS417
(TC)9CGCATATACATACATAAATTCTTTC/ 110126 5 0.72 6
0.78TCAACATCTCACCGAAGCTG45 EPMS418 (CA)10ATCTTCTTCTCATTTCTCCCTTC/
178210 8 0.84 12 0.88TGCTCAGCATTAACGACGTC46 EPMS419
(AAT)6TTCAGGTGCAGGTATCATCG/ 224248 2 0.46 9
0.78GGGTACTTGTCCATTTATCCAG47 EPMS420 (CCG)9GCATAATCGGAAAATGACGTG/
110116 2 0 4 0.55CAGCCAACTCCGAAAGCTAC48 EPMS421
(CCG)6ATCTATTTTCCTCCGGCGAC/ 236258 3 0.29 6
0.77CGGTAAGCTGCCTTGATCTC49 EPMS424 (CCT)6TCTTTTCTCTCCACCCCATG/
148160 1 0 5 0.53TTGGGCATCTTTTTCAAAGG50 EPMS426
(AT)15GAGGAAACACTCTCTCTCTCTCTCTC/ 108118 4 0.60 5
0.62TCAAGAGACCCCAAATAGGG51 EPMS427 (AAT)6(AAG)
GCTCCTCATTAGTAGCCCCC/ 113131 2 0.41 7 0.75TCTTCATCACACCTCTGTGGAC52
EPMS428 (GAT)6GGCAACCCCAATGTTGTAAC/ 318327 1 0 4
0.53CAACTCCAAACCCCTTAGGC53 EPMS429 (ATC)8 ATACTAAGGCGTTTGGTCGG/
136180 3 0.26 7 0.66ACAATTGGGGATGCAGAAAG54 EPMS430 (GAA)5ACA(GAA)
CCAACACCAAGAAATTGACG/ 255258 1 0 2 0.49CCACTTGTGACCCATTAGGG55
EPMS438 (ATT)4(TTA)2 GCTTTTTGGCAGTTTCGTTC/ 320332 1 0 5
0.46TTCCCCAGTTCACTCCTAGC56 EPMS439 (TC)6. . .(TC)7
ACTAAAGGATCCCCCGGG/ 246250 2 0.41 2 0.35CTCCCACCTGGACTCATCTG57
EPMS440 (CT)7 ACGAGGCGCCCTCTCTC/ 178192 1 0 2
0.49GAGTCCAAACTGAAGCTGCC58 EPMS441 (AG)11 GCACGAGGAAAGAGAGAGACATAG/
116124 5 0.74 5 0.82TCAACGGATTCAGTCTTCCC59 EPMS443
(TG)13imp(TA)4GGTTTTCTCACAACTTCGGC/ 190196 2 0.34 6
0.58TTGCAAAATATATCAACGCG60 EPMS446
(CAA)AG(CAA)6GAGGCCTTCTGAACCAAACC/ 328338 2 0.50 3
0.56Nagyetal.677#2007NRCCanadaTable 5 (continued).Capsicum
annuum(14 genotypes)Capsicum spp.(33 genotypes)No. Code Repeat
Forward primer / reverse primer (5?3)Allele sizerange (bp) No. of
alleles PICa No. of alleles PICcTTGTAAAGCCTTGGAGGTGG61 EPMS448
(TAA)7 GGGACGTATTTTCGAAGAGG/ 127161 1 0 8
0.76CTTCGCCTTGTTGACTAGGG62 EPMS449 (TTA)7impGCGGAGCTCACAATAAGGAG/
134147 1 0 3 0.17TGACCCAATGCTCTTCTATGC63 EPMS451
(AT)9CGTAAGACTTTACAGTGAGAGAAATC/ 166184 3 0.36 7
0.80ATTCCAGTGTCGCCGACTAC64 EPMS472 T16ATTGTGATAGCAACCCCTGG/ 289320
3 0.61 6 0.80CACAGATGAGGGCACAAATG65 EPMS480
C12A7AGGAACGGCAGTCTTGCTAG/ 241255 4 0.69 6
0.69GATGCTAGGTCTGGATTCCTG66 EPMS490 T17TTGAGGAAACGTGTGCTGAG/ 216222
2 0.49 3 0.64CGGCATTATTTCATAAGGTGG67 EPMS492
A7CA16CTCTCCAATCCTTCCCTTCC/ 187202 1 0 3
0.46GAAGAAGAAGCTAGGTTTGTTTCG68 EPMS497 G14TACACACACCATCGGGAAAG/
236248 2 0.50 7 0.78CAGTTTAGCCGAGTTTTCCG69 EPMS501
T20AATCCTCCAAATCCACCCTC/ 166180 4 0.69 10
0.88ATTCGATTGCTTGCTCCTTG70 EPMS507 A30CGGGCAGGTGCTATTATAAAAC/
196254 3 0.07 3 0.56CGGCCGAGGTACAAGCC71 EPMS514
A18GCCCATTATGACTGCACATG/ 251267 2 0.13 4
0.73CACCAAATATTCCCTGAAAGG72 EPMS538
(CAT)4imp(CAC)2(CAT)3AAGTTGCTAACGGTGCTTGG/ 118119 2 0.13 2
0.50TGAAGTTTCAGGAGCTGCTTC73 EPMS539 (TC)11 AGTGGGTTTCCTATTTGGGC/
241254 2 0.46 6 0.80CCATGAGCTGTGTTTGATGG74 EPMS540 (TGG)GG(TGG)5
TGCTATTCCAGGTAAAGCCG/ 188206 2 0.13 4 0.69TGTCGTGCGAGTCAGTCAG75
EPMS542 (TC)10 ATCCACTTCCCCATTATCCC/ 168228 4 0.68 10
0.89TGGATGATCGAGTTGACTGG76 EPMS543 (TC)4(CT)13 CTCTGCCCTCCTCAACCC/
101111 1 0 6 0.72AAAATATGGTCGGAGATCCG77 EPMS546 (ACC)7imp
AGATAATCATGGCGGCTCAG/ 219230 2 0.46 5 0.68ATCTGATGATGCCACAGCTG78
EPMS547 (AG)7 TCCTCCACTCAGCTTTCCTC/ 95108 1 0 3
0.55TTTCCTTTTGATGAATCAGGATC79 EPMS549
(ACC)4TC(ACC)4ACCAATGGATTGAATCCACG/ 150160 1 0 4
0.58AGGGCAGTTGTAGCCACTTG80 EPMS554 (TC)6T8CATCATTTCTCCCCAATTCC/
209199 1 0 6 0.60678GenomeVol.50,2007#2007NRCCanadadancy within
this repeat class was calculated to be
1.37.Thisleadstotheconclusionthat frequencyestimateswerenot
substantiallybiasedbyredundancyforthisrepeat
type.Whilethisestimationmight not
bepreciselythesamewithotherrepeats,datafromotherplants(Thieletal.2003)
sug-gest no significant differencesfor other repeattypes.Analysis
of microsatellite polymorphismsAfter inspection of all positive
microsatellite-containingsequences,411 couldbe chosenfor
primerdesign and func-tional testing. One hundred and fifty-four
markers
originatedfromgenomiclibraries(GPMSmarkers)and257originatedfrom
database sequences (EPMS markers). In all, 147markers
(61fromgenomic libraries and86fromdatabasesequences) showed
specific and scoreable
PCRproductsanddetectedpolymorphismsbetweenatleast2membersofthe
secondarytest panel consistingof 33Capsicumgeno-types.
(SeeTable3for theresultsof thefunctional
tests.)Inthemajorityofcases, thePCRproduct sizewasingoodconcordance
with the predicted size. Five EPMSmarkers(EPMS342, EPMS349,
EPMS353, EPMS369, and EPMS413)producedampliconsthat
were70to100bplargerthanex-pected, probably because of the presence
of introns.EPMS490 produced fragments that were 50 bp
shorterthanpredicted. Therepeat types, allelesizeranges,
andin-formation contents of the informative GPMS and EPMSmarkers
are shownin Table 4 and Table 5, respectively.(Primer data for the
non-informative EPMS and GPMSmarkers can be obtained fromthe
authors upon request.)Although GPMS markers detected more alleles
(7.28 2.68, on average) than EPMS markers (5.86 2.00, onaverage) in
the full set of investigated Capsicumgeno-types,
themeanPICcvalues(basedonallele-sizevariationsof
33Capsicumgenotypes) of the 2marker classes
weresimilar(0.680.13and0.670.12, respectively).
These-lectedinformativemarkersshowedalowlevel ofpolymor-phism
within C. annuum: with low mean
intraspecific(PICa)values(0.27forGPMSmarkersand0.30forEPMSmarkers),
41%of the GPMS markers and 28%of theEPMS markers detected only one
allele within the 14C. annuumgenotypes of thetest panel (seeFig.
1Basanexample). On the other hand, some highly informativemarkers
exhibited relatively high allelic variations withinC. annuum(Fig.
1A). Correlation coefficient calculationsbetween PICcvalues andcore
repeat length resulted inrvaluesof 0.31(P