A set of conserved PCR primers for the analysisof simple sequence repeat polymorphisms inchloroplast genomes of dicotyledonousangiosperms

Kurt Weising and Richard C. Gardner

Abstract: Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice,and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues insuch repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotiderepeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)nrepeats (n $ 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed andused to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCRproducts from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species.Levels of interspecific polymorphism within the generaNicotiana, Lycopersicon(both Solanaceae), andActinidia(Actinidiaceae) proved to be high, while intraspecific variation inNicotiana tabacum, Lycopersicon esculentum, andActinidia chinensiswas limited. Sequence analysis of PCR products from three primer pairs revealed variable numbersof A, G, and T residues in mononucleotide arrays as the major cause of polymorphism inActinidia. Our resultssuggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplastvariation in angiosperms.

Key words: genetic markers, chloroplast genome, microsatellites, consensus primers, angiosperms.

Résumé: De courts séries de répétitions mononucléotidiques sont présentes dans les génomes chloroplastiques desplantes supérieures. Chez le soya, le riz et le pin, la PCR réalisée à l’aide d’amorces adjacentes à ces microsatellites amontré que le nombre de résidus A ou T est variable parmi des taxons très apparentés. Ici, les auteurs décrivent desamorces qui permettent d’étudier la variation au niveau des microsatellites de l’ADN chloroplastique chez lesangiospermes, un groupe d’organismes pour lequel l’information présente dans les bases de données est limitée. Untotal de 39 répétitions (A)n et (T)n (n $ 10) ont été identifiées dans le génome chloroplastique du tabac et lesséquences nucléotidiques de ces 39 régions ont été alignées avec des séquences orthologues trouvées dans les bases dedonnées. Des paires d’amorces consensuelles ont été préparées et utilisées pour amplifier l’ADN total d’un ensemblehiérarchique d’angiospermes. Les dix paires d’amorces ont permis d’obtenir des produits PCR chez des membres dessolanacées et huit des dix paires se sont avérées fructueuses chez la plupart des autres angiospermes. Le degré depolymorphisme interspécifique à l’intérieure des genresNicotiana, Lycopersicon(tous deux des solanacées), etActinidia (actinidiacées) s’est avéré élevé tandis que la variation intraspécifique chez leNicotiana tabacum, leLycopersicon esculentumet l’Actinidia chinensisétait limitée. Le séquençage des produits PCR obtenus à l’aide detrois paires a révélé que le nombre variable de résidus A, G et T était la principale cause du polymorphisme chezl’ Actinidia. Ces résultats suggèrent que des amorces universelles permettant d’amplifier des microsatellites peuventservir d’outils généraux pour étudier la variation chloroplastique chez les angiospermes.

Mots clés: marqueurs génétiques, génome chloroplastique, microsatellites, amorces consensuelles, angiospermes.

[Traduit par la Rédaction]

Weising and Gardner 19

Genome 42: 9–19 (1999) © 1999 NRC Canada

9

Corresponding Editor: B. Golding.

Received March 18, 1998. Accepted July 16, 1998.

K. Weising1 and R.C. Gardner. Centre for Gene Technology, School of Biological Sciences, University of Auckland,Private Bag 92019, Auckland, New Zealand.

1Author to whom all correspondence should be addressed: Priv. Doz. Dr. Kurt Weising, Plant Molecular Biology,Biozentrum, N200, Johann Wolfgang Goethe University, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany(e-mail: weising@em.uni-frankfurt.de).

The conserved nature of the chloroplast genome in higherplants (Wolfe et al. 1987) has some practical implicationsfor genetic research. Most importantly, the high degree ofsequence conservation has facilitated the use of heterologoushybridization probes and polymerase chain reaction (PCR)primers in unrelated species, thereby circumventing the needto clone chloroplast DNA (cpDNA) from each and everyspecies under study (Olmstead and Palmer 1994). UniversalPCR primer pairs have been constructed on the basis of con-served coding sequences of cpDNA genes and used to am-plify the DNA located between the primer binding sites(Taberlet et al. 1991; Demesure et al. 1995; Dumolin-Lapegue et al. 1997). Direct sequencing or restriction analy-sis of the PCR products often yielded informativepolymorphisms, which were exploited to study taxonomicand phylogenetic relationships at various systematic levels(Arnold et al. 1991; Cipriani et al. 1995; Testolin andCipriani 1997; Wolfe et al. 1997).

An undesirable consequence of the extensive cpDNA se-quence conservation is the difficulty to discriminate betweenclosely related chloroplast genomes. While intraspecificcpDNA restriction site variation has been reported for manyspecies (reviewed by Soltis et al. 1992), the magnitude ofsuch variability is generally low. The need to test many re-striction enzymes in order to detect a single polymorphismmakes the search for intraspecific cpDNA variation quitecumbersome, even when assisted by PCR with universalprimers. Recently, a new cpDNA marker system has beenestablished that is based on the occurrence of a certain typeof “microsatellite” in the chloroplast genome (Powell et al.1995a, 1995b). Microsatellites, also called “simple sequencerepeats,” are abundant polymorphic elements of eukaryoticnuclear genomes and consist of tandemly reiterated, shortDNA sequence motifs (Wang et al. 1994; Field and Wills1996). Size variation of microsatellites is due to a variablerepeat copy number and can be visualized by PCR with pairsof flanking primers and electrophoretic separation of the am-plification products. Nuclear microsatellites are oftenmultiallelic within and among populations, inherited in acodominant fashion, and fast and easy to type. They havetherefore become the marker system of choice for geneticmapping, population genetics, and DNA profiling in plants(Powell et al. 1996a; Weising et al. 1998).

Database surveys have shown that microsatellites arecomparatively rare in organellar DNA (Wang et al. 1994). Infact, the only type of simple repeat found in the chloroplastgenome of higher plants is made up of short poly(A) orpoly(T) tracts, with maximum sizes of about 20 bp. Never-theless, a number of studies employing flanking PCR prim-ers have shown that such mononucleotide stretches arepolymorphic among different species and accessions ofGlycine (Powell et al. 1995b, 1996b), Oryza (Provan et al.1996, 1997), and several gymnosperms (Powell et al. 1995a;Cato and Richardson 1996; Vendramin et al. 1996). Wher-ever the PCR products were analyzed by DNA sequencing,the length variability was found to be due to a variable num-ber of A or T residues. These observations suggest that sim-ple sequence repeats in chloroplasts may provide a generalmarker system for evaluating the genetic structure of plant

populations and for studying the mode of chloroplast inheri-tance.

As with nuclear microsatellites, a more widespread use ofthe approach is currently limited by the need of sequencedata for primer construction. In all studies published so far,primer pair sequences for the amplification of chloroplastmicrosatellites were deduced from database entries. We areinterested in applying chloroplast markers for inheritancestudies in wild and cultivated kiwifruit (Actinidia deliciosaand relatives), where database information is limited. Oneway to approach this problem would be the construction ofconsensus primer pairs designed to amplify chloroplastmicrosatellites in many different plant species. Such primerpairs would have to meet three criteria. First, their target se-quences need to be sufficiently conserved to generate a frag-ment from unrelated species. Second, the amplificationproducts should be polymorphic, e.g., by harbouring a vari-able mononucleotide repeat. Third, the products should besufficiently short to allow single-base resolution on sequenc-ing gels.

Here we describe a set of 10 consensus primer pairs basedon multiple alignment of mononucleotide repeat-flanking re-gions in cpDNA from several mono- and dicotyledonousplant species. We show that 8 of the 10 primer pairs areubiquitously applicable across dicotyledonous angiosperms,and reveal intra- and interspecific cpDNA polymorphismswithin the generaNicotiana, Lycopersicon,and Actinidia.

Plant materialsAll Actinidia plant material originated from Hort Research or-

chards, Kumeu and Te Puke, New Zealand. Tomato species andcultivars were derived from the Botanical Garden, University ofFrankfurt, Germany. A set of 12N. tabacumcultivars was derivedfrom Nelson Research Centre, Nelson, New Zealand. Origins ofall other plant material used in the present study are summarizedin Table 2. Leaves were either used fresh, or frozen in liquid ni-trogen, and stored at –80°C.Pinus radiataDNA was a gift fromT. Richardson, Forest Research Institute, Rotorua, New Zealand.

DNA isolationGenomic DNA was isolated from young leaves of different

angiosperm species using various modifications of the cetyltrimethylammonium (CTAB) procedure (Weising et al. 1995,1996). DNA concentrations were determined electrophoreticallyagainst known amounts ofλ DNA as standards. For PCR, DNAsamples were adjusted to a concentration of 20 ng/µL.

Database studiesThe Genetic Computer Group (GCG) software package

(Devereux et al. 1984) was used for all DNA sequence analysesexcept for the homology searches (see below). Initially, the fullysequenced tobacco chloroplast genome (Shinozaki et al. 1986) wasscreened for the presence of mononucleotide runs withn $ 10 us-ing FINDPATTERNS. For each candidate, 400 bp of sequence en-compassing the repeat were then excised using SEQED, andscreened for homologies in the EMBL and GenBank databases us-ing BLASTN (Altschul et al. 1990). Finally, the PILEUP softwarewas applied to generate multiple alignments of homologous se-quences with the respective tobacco cpDNA region.

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10 Genome Vol. 42, 1999

Primer designFrom the alignment information, 10 loci were selected for con-

sensus primer pair construction using the program Cprimer (writ-ten by G. Bristol and R.D. Anderson, School of Medicine,University of California, Los Angeles, Cal.) which is availablefrom the Internet (see Results section for the criteria for candidateselection). Primer pairs were checked for the absence ofself-annealing, dimer and hairpin formation capacities. Primerspacing was chosen to result in PCR products with an expectedsize range of 80–160 bp in tobacco. Primers fulfilling all criteriawere purchased from Gibco-BRL. A set of 20 additional primerpairs (MapPairs) specific forPinus thunbergii chloroplastmicrosatellites (Vendramin et al. 1996) was purchased fromResearch Genetics.

Microsatellite analysis using radioisotopesRadioactive PCR was performed in 10µL volumes using a

Techne PHC-3 thermal cycler. Each reaction contained 20 ng oftemplate DNA, 2.5 mM MgCl2, 0.5 µM each of forward and re-verse primer, 0.2 mM each of dATP, dGTP, and dTTP, 0.02 mM ofdCTP, 0.04µL of 3000 Ci/mmole α[32P]dCTP or α[33P]dCTP,10 mM Tris–HCl at pH 8.3, 50 mM KCl, and 1 unit AmpliTaqDNA polymerase (Perkin Elmer). Reactions were overlayed withtwo drops of mineral oil. The PCR regime generally followed theconditions described by Vendramin et al. (1996), but with a lowerannealing temperature. After an initial denaturation at 94°C for5 min, PCR was performed for 30 cycles, each consisting of 94°Cfor 1 min, 50°C for 1 min, and 72°C for 1 min. Final extensionwas at 72°C for 8 min. PCR products were mixed with 1 vol. of97.5% (v/v) formamide, 10 mM EDTA at pH 7.5, 0.3% (w/v)xylene cyanol, 0.3% (w/v) bromophenol blue, and were denaturedat 95°C for 3 min. Aliquots of each sample were electrophoresedon denaturing polyacrylamide gels (6% acrylamide–bisacrylamide(19:1), 8 M urea in Tris–borate–EDTA buffer, pH 8.3) at constantpower (55 W) for 2–3 h. Sequencing reactions of M13mp9 sin-gle-stranded DNA (Boehringer Mannheim) were used as molecularweight standards. The gels were dried at 80°C for 45 min, and ex-posed to Kodak XR5 film without intensifying screens for 3–24 h.Bands were scored by inspection, with the allele sizes calculatedfrom the most intense band.

Microsatellite analysis using fluorescent dyesFluorescent PCR was performed in 10µL volumes using a

GeneAmp 2400 thermocycler (Perkin Elmer). Fluorescent dUTPswere purchased from Perkin Elmer. Each reaction contained 20 ngof template DNA, 2.5 mM MgCl2, 0.5µM each of forward and re-verse primer, 0.2 mM of each dNTP, 10 mM Tris–HCl at pH 8.3,50 mM KCl, 1 unit AmpliTaq DNA polymerase (Perkin Elmer)and either 1µM dUTP[R6-G] (green), 1µM dUTP[R110] (blue),or 4 µM dUTP[TAMRA] (yellow). The same PCR regime wasused as described above. PCR products containing each of threedifferent fluorochromes were pooled, and combined with a[ROX]-labelled molecular weight standard (red fluorescence).Mixed samples were diluted 1:5 or 1:10, denatured as above,and electrophoresed on denaturing polyacrylamide gels (6%acrylamide–bisacrylamide (19:1) and 8 M urea in TBE at pH 8.3)using an Applied Biosystems 373A DNA sequencer. Fragment mo-bilities were measured by real-time laser scanning, and sizes weredetermined using the ABIPRISM GeneScan and Genotyper soft-ware packages (Perkin Elmer).

Sequencing of PCR fragmentsThe PCR products from 16 primer-template combinations were

characterized by direct DNA sequencing. Large-scale PCR wasperformed in 100µL volumes as described above, but omitting flu-orescent and radiolabelled nucleotides. An aliquot of the PCR

product was checked by agarose electrophoresis, and the remainderwas purified through a High Pure PCR Purification Kit(Boehringer Mannheim). Double-stranded sequencing was per-formed by the dideoxynucleotide chain termination method usingan Applied Biosystems 373A DNA sequencer. Both strands weresequenced for each product. Sequences have been deposited in theDDJB nucleotide sequence database (accession numbersAB006089–AB006101). The LINEUP and PILEUP programs ofthe GCG package were used to generate multiple sequence align-ments (Devereux et al. 1984).

Design of consensus primer pairs that flank chloroplastmicrosatellites

The initial objective of the present study was to developprimer pairs that detect cpDNA polymorphisms among culti-vated kiwifruit (Actinidia deliciosa) and related species. Re-cently, Cato and Richardson (1996) have reported successfulamplification of Actinidia chinensiscpDNA, using two outof five primer pairs derived from mononucleotide repeat-flanking regions ofPinus thunbergii, a gymnosperm. Basedupon this observation, we tested a set of 20 primer pairs de-signed for the amplification of pine chloroplast micro-satellites (Vendramin et al. 1996) with total DNA fromA. chinensis, usingPinus radiataDNA as a positive control.All primer pairs produced bands withPinus DNA as ex-pected, but only one pair reproducibly yielded a single PCRproduct in the expected size range withA. chinensisDNA(not shown). All other primers either revealed no product, asmear, or a multilocus pattern when reduced stringency con-ditions were applied. Analyzing the only functional primerpair on a set ofA. chinensisaccessions on a sequencing gelshowed no size variation. The conclusion from these experi-ments was that conservation of mononucleotide repeat-flanking regions between gymno- and angiosperms is quitepoor.

We therefore decided to design a set of primer pairs solelybased on angiosperm cpDNA. The completely sequencedchloroplast genome ofNicotiana tabacum(Shinozaki et al.1986) was used as a starting point. Screening the tobaccocpDNA for mononucleotide arrays withn $ 10 yielded 39poly(A) and poly(T) repeats, whereas extended poly(G) orpoly(C) stretches were absent (see also Powell et al. 1995b).Since eight of the repeats formed four closely adjacent pairs,and two repeats were located within the identical, invertedrepeat regions, the total number of useful candidatesdropped to 33. EMBL and GenBank databases werescreened for cpDNA sequences homologous to each candi-date region, and orthologous regions were aligned to 400 bpsegments encompassing the tobacco repeats. Reasonablealignments were possible for 25 of the 33 candidates (align-ments are available from the authors upon request).

Two criteria were applied to select the most promisingcandidates for primer pair design. First, only those loci wereconsidered where putative primer target regions flanking themononucleotide repeat were sufficiently conserved. In thisrespect, particular attention was given to the primer 3′ end.Second, the sequence internal to the primer binding sitesshould harbour a ± long poly(A) or poly(T) tract not only intobacco, but also in other species (which was the case inabout half of the candidates). We reasoned that the first cri-

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Weising and Gardner 11

terion would help to maximize the transportability of prim-ers across taxa, while the second criterion would increasethe chance of size variation. Primer pairs were designed for10 candidates which met both criteria best. We named thisset of primers: consensus chloroplast microsatellite primers(ccmp), ccmp1 to ccmp10. Their sequences, calculatedTmvalues, as well as the size, type, and location of the corre-sponding microsatellite repeat in the tobacco cpDNA, aresummarized in Table 1. From the sequence alignments, themost promising candidate was ccmp10, amplifying therpl2–rps19intergenic region, with poly(A) tracts of variablesize in almost all species examined (see also Goulding et al.1996).

Screening of consensus chloroplast microsatelliteprimers with a set of angiosperms

All 10 primer pairs were tested with a hierarchically struc-tured DNA template set, consisting of 13 samples from thenightshade family (including the generaNicotiana,Lycopersicon, Petunia, andSolanum), five Actinidia species,six other dicotyledons from various families, and threemonocots (Table 2). Total leaf DNA was amplified in thepresence of radioactive dCTP, and the PCR products wereseparated on sequencing gels and visualized by auto-radiography. A standard PCR protocol (with an annealingtemperature of 50°C) was used for all primer pairs, irrespec-tive of the calculatedTm values (Table 1). The results ob-tained with two primer pairs are shown in Fig. 1. The allelesizes obtained with all primer pairs across all the species arecompiled in Table 2.

The results can be summarized as follows: (i) All primerpairs produced a fragment of the expected size fromNicotiana tabacum. All other Solanacean species were alsoamplified, with the exception of ccmp8 which failed to pro-duce a product with two templates. Moreover, 8 of the 10primer pairs (i.e., ccmp1–7 and ccmp10) also generatedproducts for the majority of tested species from otherdicotyledonous angiosperm families, while amplification ofmonocotyledons was somewhat less efficient. (ii ) PCR prod-ucts showed considerable size variation, not onlyamongtheinvestigated genera and families, but alsowithin genera.Alleles were often separated by steps of 1 bp, which is con-sistent with a variable number of A or T residues in a mono-nucleotide repeat. (iii ) No intraspecific variation was foundin this series of experiments. Also, theActinidia deliciosaandA. chinensissamples were always identical, which sup-ports the close relationship of these two species (Ciprianiand Morgante 1993; Atkinson et al. 1997). With one excep-tion (ccmp2), identical product sizes were also observedbetweenNicotiana tabacumand N. sylvestriswhich is con-sidered to be a progenitor of the allotetraploid cultivated to-bacco (Gray et al. 1974).

The well-known “stuttering” phenomenon, which usuallyoccurs upon amplification of mono- and dinucleotide-typemicrosatellites, was observed with all primers. This effect isthought to be a consequence of polymerase slippage duringreplication (Litt et al. 1993). Comparison of band sizes tothe known tobacco sequence showed that the band second tothe top is the “correct” band among the cluster of bandswhich were separated by one base pair. Differences between

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Position Repeat in Primers deduced from multiple Tm (°C) Size inCode (bp) Location tobacco sequence alignment Wal Cpr tobacco (bp)

ccmp1 3801 trnK intron (T)10 5’-CAGGTAAACTTCTCAACGGA-3’5’ -CCGAAGTCAAAAGAGCGATT-3’

5858

53.357.3

139

ccmp2 8609 5’ totrnS (A)11 5’-GATCCCGGACGTAATCCTG-3’5’-ATCGTACCGAGGGTTCGAAT-3’

6060

58.058.6

189

ccmp3 10 075 trnG intron (T)11 5’-CAGACCAAAAGCTGACATAG-3’5’-GTTTCATTCGGCTCCTTTAT-3’

5856

51.353.9

112

ccmp4 12 872 atpF intron (T)13 5’-AATGCTGAATCGAYGACCTA-3’5’-CCAAAATATTBGGAGGACTCT-3’

6058

55.356.1

126

ccmp5 16 95016 977

3’ to rps2 (C)7(T)10

(T)5C(A)11

5’-TGTTCCAATATCTTCTTGTCATTT-3’5’-AGGTTCCATCGGAACAATTAT-3’

6258

55.055.4

121

ccmp6 45 119 ORF 77–ORF 82intergenic

(T)5C(T)17 5’-CGATGCATATGTAGAAAGCC-3’5’-CATTACGTGCGACTATCTCC-3’

5860

52.752.5

103

ccmp7 57 339 atpB–rbcLintergenic

(A)13 5’-CAACATATACCACTGTCAAG-3’5’-ACATCATTATTGTATACTCTTTC-3’

5658

45.144.5

133

ccmp8 71 563 rpl20–rps12intergenic

(T)6C(T)14 5’-TTGGCTACTCTAACCTTCCC-3’5’-TTCTTTCTTATTTCGCAGDGAA-3’

6058

52.953.9

77

ccmp9 74 060 ORF 74b–psbBintergenic

(T)11 5’-GGATTTGTACATATAGGACA-3’5’-CTCAACTCTAAGAAATACTTG-3’

5456

45.144.2

98

ccmp10 86 694 rpl2–rps19intergenic

(T)14 5’-TTTTTTTTTAGTGAACGTGTCA-3’5’-TTCGTCGDCGTAGTAAATAG-3’

5658

53.353.7

103

Table 1. Size and position of 10 tobacco cpDNA microsatellites selected for the construction of consensus primerpairs ccmp1–ccmp10.Tm values were calculated by the Wallace rule (Thein and Wallace 1986), or according to the Cprimerprogram (based on an algorithm described by Breslauer et al. 1986). Degenerate positions are Y (= C or T), B (= G, C, or T)and D (= A, T, or G).

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Size of amplification products (bp)

Lane Species Family ccmp1 ccmp2 ccmp3 ccmp4 ccmp5 ccmp6 ccmp7 ccmp8 ccmp9 ccmp10

a Nicotiana tabacumcv. Petit Havana (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103b N. tabacumcv. Virginia (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103c N. tabacumcv. Atropurpurea (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103d N. silvestris(FRA) Solanaceae 139 188 112 126 121 103 133 77 99 103e N. acuminata(FRA) Solanaceae 134 188 113 123 119 101 129 — 103 102f N. benthamiana(AUK) Solanaceae 139 189 109 128 118 100 130 77 101 109g Lycopersicon hirsutum(FRA) Solanaceae 140 189 114 123 119 93 133 65 101 110h L. peruvianum(FRA) Solanaceae 143 188 113 123 118 93 133 65 96 111i L. esculentumvar. finiens (FRA) Solanaceae 141 190 114 122 119 93 134 65 101 109j L. esculentumcv. Haubner’s Vollendung (FRA) Solanaceae 141 190 114 122 119 93 134 65 101 110k L. esculentumcv. UC82B (HORT) Solanaceae 141 190 114 122 119 93 134 65 101 110l Petunia hybrida(AUK) Solanaceae 138 193 108 125 118 96 131 — 104 113m Solanum tuberosum(FRA) Solanaceae 140 188 115 124 119 93 133 66 98 110n Actinidia arguta(HORT) Actinidiaceae 131 206 114 138 89 102 135 — — 91o A. polygama(HORT) Actinidiaceae 132 208 114 137 98 102 133 — — 91p A. chrysantha(HORT) Actinidiaceae 131 210 107 138 98 102 132 — — 92q A. chinensis(HORT) Actinidiaceae 130 209 107 138 98 102 131 — — 91r A. deliciosavar. coloris (HORT) Actinidiaceae 130 209 107 138 98 102 131 — — 91s Brassica oleracea(AUK) Brassicaceae 139 166 103* 128* 119 93 140 — 101 96t Sinapis alba(AUK) Brassicaceae 122 165 104 124 — 94 140 — — 96u Arabidopsis thaliana(AUK) Brassicaceae 140 158 103 124 — 96 140 — — 95v Pisum sativum(AUK) Fabaceae 139 234 93 115 109 111 146 — 101* ~230w Metrosideros excelsa(AUK) Myrtaceae 139* 211 119 135 107 103 151 — 101 98x Malus domestica(HORT) Rosaceae 127 196 100 126 122 106 132 — — 113y Hordeum vulgare(AUK) Poaceae 139 182* — ~220 145 98 130 — 101* 96*z Lolium multiflorum (AUK) Poaceae 139 186* — — 160 95 130 — 101 96*β Cordyline australis(AUK) Agavaceae 139 187* 89 168 77 100 130* — 101 >300

Note: Abbreviations of plant material origins are: FRA (Botanical Garden, University of Frankfurt, Germany), AUK (University of Auckland, New Zealand), and HORT (Hort Research Orchards,Kumeu and Te Puke, New Zealand).

Table 2. Allele sizes of amplification products from a hierarchical set of angiosperm species, generated by primers ccmp1–ccmp10. Radiolabelled DNA fragments wereseparated on sequencing gels and visualized by autoradiography. An asterisk indicates that more than a single band was amplified by the respective primer–templatecombination. In this case, the allele size of the strongest band is given.

samples were easily recognized, because the whole clusterswere then shifted relative to each other (see Fig. 1).

Inter- and intraspecific variation in Nicotiana,Lycopersicon, and Actinidia

To test whether the failure to detect intraspecificpolymorphisms was due to the limited number of accessionsexamined, we next analyzed a broader set of samples, in-cluding 12 additional tobacco varieties, two moreNicotianaspecies (N. otophoraandN. glutinosa), six additional tomatocultivars, the wild tomato species Lycopersiconpimpinellifolium, and 22 accessions belonging to 10 differ-ent Actinidia species (including all samples from Table 2plus A. guilinensis, A. hemsleyana, A. macrosperma,A. melanandra, andA. setosa). To achieve a higher samplethroughput, PCR products from different primer/template

combinations were pooled and analyzed by automated fluo-rescence detection (Ziegle et al. 1992; Diwan and Cregan1997). Some samples were evaluated by both fluorescenceand radioactivity to test the comparability of both ap-proaches. Fragment sizes automatically called by theGenescan and Genotyper softwares were in a range of ± 0.6bp of those detected by autoradiography. This is similar inmagnitude to variation found in studies on nuclearmicrosatellites (Diwan and Cregan 1997). Provided that flu-orescence gels were run at high resolution and peaks wereevaluated carefully, consistent size differences between frag-ments were obtained with both techniques.

Allele sizes revealed by the fluorescence approach aresummarized in Table 3. The full data sets obtained for indi-vidual samples are available from the authors upon request.Again, there was considerableinterspecific variation within

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14 Genome Vol. 42, 1999

Fig. 1. Radioautographs of amplification products from a hierarchical set of angiosperm species, generated by primers ccmp7 (top)and ccmp2 (bottom) and separated on sequencing gels. Numbering of lanes is according to Table 2 (see text for details). M13mp9sequencing reactions served as molecular weight markers.

Nicotiana, with 3 to 5 different band sizes among six spe-cies. However,intraspecific variation amongN. tabacumcultivars was limited, with two notable exceptions. First,primer pair ccmp2 yielded alleles of 189 and 190 bp, withsimilar frequency among the individual cultivars ofN. tabacum and the diploid N. sylvestris. Second, thechloroplast haplotype ofN. tabacumKentucky 34 was com-pletely identical to that ofN. glutinosa(see Discussion). Intomato, most alleles were shared betweenLycopersiconesculentumand its putative progenitor,L. pimpinellifolium.Intraspecific polymorphisms were observed with primerpairs ccmp9 and 10 only, both detecting two alleles each(Table 3).

Analysis of theActinidia samples revealed between oneand five size variants among the 10 species examined(Table 3). Each species was characterized by a uniquehaplotype, except forA. deliciosawhich was identical to themost common haplotype found inA. chinensis(see below).Alleles of different species often differed by steps of 1 bp(see Table 2), but larger differences were also observed (e.g.,107, 114, 115, and 122 with ccmp3). Similar to the situationin tobacco and tomato, the extent of intraspecific polymor-phism was limited. Variation was only observed with primerpairs ccmp3 and ccmp10, which detected two alleles eachamong the eight diploidA. chinensisaccessions examined.These allowed discrimination of three haplotypes. The morecommon allele of each pair was also present in tetraploidA.chinensisand hexaploidA. deliciosavarieties.

Sequence analysis of cpDNA amplification productsfrom Actinidia species

To credibly examine whether the observed size differ-ences were due to microsatellite variation, or othermutational events, we sequenced the PCR products obtainedfrom 3 primer pairs and 5Actinidia species, and alignedthese sequences to the corresponding tobacco sequence de-rived from the database (Fig. 2). The alignments demon-strate that variable numbers of mononucleotide repeats arein fact the major cause of polymorphism amongActinidiaspecies. Primer pairs ccmp1, 2, and 7 generated a total of 3,4, or 5 variants, respectively. The three ccmp1 variants sim-ply differ by the number of T’s (T10 vs. T11 vs. T12), whilethe other loci are somewhat more complex. In caseof ccmp7, runs of bothA andG residues were found respon-sible for the observed variability, reminiscent of the situationin a soybean tRNAMet gene (Powell et al. 1995b). In allthree cases,A. chinensisand A. deliciosaalleles were com-pletely identical. Quite unexpectedly, the mononucleotide re-peats responsible for the polymorphism amongActinidiaspecies were not necessarily those originally selected for thealignment (underlined in Fig. 2). Instead, variability wasfound within adjacent shorter repeats in two of three regionssequenced.

We have applied a consensus PCR primer concept to thestudy of small length polymorphisms caused by variablenumbers of A and T residues in mononucleotide repeats incpDNA (Powell et al. 1995a, 1995b). A serious challengefor primer design was that PCR products had to be suffi-

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Weising and Gardner 15

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ciently short (<200 bp) to ensure discrimination of one-basedifferences on sequencing gels. In contrast to previouslypublished “universal primer” strategies (for example,Taberlet et al. 1991; Demesure et al. 1995; Dumolin-Lapegue et al. 1997), at least one primer of each pair had to

be targeted to noncoding cpDNA. Despite the comparativelylow base-substitution rate of cpDNA (Wolfe et al. 1987),non-coding regions in general are not sufficiently conservedto guarantee primer transferability between gymno- and angio-sperms (Cato and Richardson 1996, this study). Insufficient

© 1999 NRC Canada

16 Genome Vol. 42, 1999

ccmp 11 50

A. chinensis TTCTCTATCC TCTCTTTTTC CATTTAATGG GTTTA..... TGTTCGTTATA. chrysantha TTCTCTATCC TCTCTTTTTC CATTTAATGG GTTTA..... TGTTCGTTATA. arguta TTCTCTATCC TCTCTTTTTC CATTTAATTG GTTTA..... TGTTCGTTATA. polygama TTCTCTATCC TCTCTTTTTC CATTTAAGTG GTTTA..... TGTTCGTTATN. tabacum TTCTCTATCA TCTCTTTTTT TTTTCGTTTC GTTTAATTGG TCTATGTTAT

51 100A. chinensis AGGAGAAGAA GACGGTTAGA .AATCCTTTA..TTTTTTTT TT GCAACCCAA. chryeantha AGGAGAAGAA GACGGTTAGA .AATCCTTTA.TTTTTTTTT TT GCAACCCAA. arguta AGGAGAAGAA GACGGTTAGA .AATCCTTTA.TTTTTTTTT TT GCAACCCAA. polygama AGGAGAAGAA GACGGTTAGA .AATCCTTTATTTTTTTTTT TTGCAACCCAN. tabacum AGTGTTATAG GATAATAAGA TGGTTAGAAA TCCTTTATTT TTTCAACCTA

ccmp 21 50

A. chinensis AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTT..AA AAAAAAAATTA. chryeantha AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTT.AAA AAAAAAAATTA. setosa AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTTAAAA AAAAAAAATTA. arguta AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTTT... ..AAAAAA TTA. polygama AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATT...AA AAAAAAAA TTN. tabacum AAAATAAAAA AGGT.....T TTTCCTTGCT TGATTTT... ..CCAATTTT

51 100A. chinensis CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGAA. chrysantha CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGAA. setosa CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGAA. arguta CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGAA. polygama CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGAN. tabacum CTTATGATTT GGTCTATTCC ACACATTTAA CTAAGAATAA GAACAAAGGA

101 150A. chinensis TTTGCAAAAT TTGAAAGAGA AATCAAATAT CAAGTCATCC AAGGAAACGGA. chrysantha TTTGCAAAAT TTGAAAGAGA AATAAAATAT CAAGTCATCC AAGGAAACGGA. setosa TTTGCAAAAT TTGAAAGAGA AATCAAATAT CAAGTCATCC AAGGAAACGGA. arguta TTTGCAAAAT TTGAAAGAAA AATCAAATAT CAAGTCATCC AAGGAAACGGA. polygama TTTGCAAAAT TTGAAAGCGA AATCAAAGAT CAAGTCATAC AAGGAAACGGN. tabacum TTTCGAAATT TGAAAAAAAA AA......A T CAAGTCATC. .....AACGG

ccmp 71 50

A. chinensis AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAAA. polygama AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAAA. chrysantha AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAAA. arguta AGGGAATTTC TTATTCTTTG AGGTTATTTC GGTATTTCGA TTCAAAAAAAN. tabacum GGGGAAGTTC TTATTATTT. AGGTTAGTCA GGTATTTCCA TTTCAAAAAA

51 92A. chinensis ..GGGGGGGT .AAAAATAAG AATTGGGTTG CGCCATATAT ATA. polygama A.GGGGGGGT CAAAAATAAG AATTGGGTTG CGCCATATAT ATA. chrysantha .GGGGGGTGT .AAAAATAAG AATTGGGTTG CGCCATATAT ATA. arguta GGGGGGGGGT AAAAAATAAG AATGGGGTTG CGCCATATAT ATN. tabacum AAAAAAAG.T AAAAAAGAAA AATTGGGTTG CGCTATATAT AT

Fig. 2. Alignment of cpDNA sequences fromActinidia species amplified by primer pairs ccmp1, ccmp2, and ccmp7 with thecorresponding sequences of theNicotiana tabacumcpDNA (Shinozaki et al. 1986). The repeat containing the length polymorphism isshown in bold, as are other nucleotide differences between theActinidia sequences. The long repeat originally targeted in the tobaccoalignment is double-underlined. AllA. deliciosasequences were completely identical to the correspondingA. chinensissequences andwere therefore omitted.

sequence conservation of non-coding primer target sitesproved to be a problem also within angiosperms, and diffi-culties with alignment reduced the number of promisingconsensus primer candidate regions from 39 to about 15.Nevertheless, the 10 ccmp pairs that were finally selectedworked well within Solanaceae. More importantly, eight ofthese 10 amplified most other dicotyledonous species, andsome primers also worked with the three monotyledons ex-amined. These results demonstrate that the construction ofuniversally applicable consensus primers from mono-nucleotide-repeat flanking regions in cpDNA is feasible. Formonocotyledons, it may be desirable to design an independ-ent consensus primer set based on the completely sequencedrice and maize chloroplast genomes.

The analysis of a hierarchical set of species and cultivarsshowed that PCR products generated by ccmp pairs werepolymorphic at various levels. As expected, fragment sizevariation increased with the phylogenetic distance betweenthe investigated taxa. The extent of variability depended onthe locus. The largest size spectrum was observed within therpl2–rps19 intergenic region amplified by ccmp10, rangingfrom 91 bp forActinidia up to more than 300 bp for the cab-bage tree,Cordyline australis. This same locus has also beenfound highly variable among Solanaceae by Goulding et al.(1996). As was also observed in rice (Provan et al. 1996),the amount of variation was not associated with the size ofthe particular poly(A) or poly(T) repeat under study. For ex-ample, the ccmp6 pair flanked the longest mononucleotiderepeat found in the tobacco cpDNA [i.e., (T)5C(T)17], butPCR products generated by this primer pair were among theleast polymorphic in all species investigated (Tables 2 and3). In nuclear microsatellites, the variability (and hence themutability) of a locus was often shown to be positively cor-related with the number of uninterrupted repeats, also inplants (e.g., Saghai-Maroof et al. 1994). The reasons for thiscontrasting behaviour are not clear, but may relate to differ-ent mechanisms of repeat generation and expansion in nucleiversus chloroplasts.

Intrageneric variation was considerable inNicotiana,Lycopersicon, and Actinidia, demonstrating the usefulnessof ccmp pairs for detecting polymorphisms below the genuslevel. However, comparatively few intraspecific poly-morphisms were found inNicotiana tabacum, Lycopersiconesculentum, and Actinidia chinensis. Much higher levels ofintraspecific chloroplast mononucleotide repeat variationwere recently reported from other plant taxa such as soybean(Powell et al. 1995b, 1996b), pine (Powell et al.1995a, Catoand Richardson 1996) and rice (Provan et al.1996, 1997).We consider it unlikely that insufficient sampling caused thepaucity of variation observed in the present study, especiallyin the case of tobacco where a world-wide collection ofcultivars was analyzed. It seems more likely that these con-trasting results are the consequence of a species-specificcomponent of variation. Restriction site analyses also dem-onstrated that cpDNA sequence divergence can vary consid-erably among species (reviewed by Soltis et al. 1992).

The low overall level of intraspecific variation among to-bacco cultivars made it quite obvious that the chloroplasthaplotype of cv. Kentucky 34 was atypical forN. tabacum,but fully compatible withN. glutinosa. This exceptional be-haviour can be explained by the breeding history of this

cultivar. About 50 years ago, extensive breeding programsin Kentucky and elsewhere were directed towards theintrogression of theN gene (conferring tobacco mosaic virusresistance) fromN. glutinosainto cultivatedN. tabacumva-rieties (Valleau 1952). Interspecific crosses between bothspecies were preferably made in one direction, whereN. tabacumserved as the pollen andN. glutinosaas the seedparent (Valleau 1952). Maternal inheritance of cpDNA, as isassumed to occur in the genusNicotiana (Scowcroft 1979)would explain the persistence of theN. glutinosacpDNAhaplotype in Kentucky cultivars.

Sequencing of amplification products from 16 allele/spe-cies combinations showed that variable copy numbers ofmononucleotide repeats were a main factor underlying PCRfragment length variation inActinidia species. At two out ofthree loci, however, the variability in repeat length was notfound in the long mononucleotide stretch selected from thetobacco cpDNA, but occurred in closely adjacent, shorter re-peats. There are at least two possible explanations for thesesurprising results. One is that poly(A) or poly(T) repeats areclustered in chloroplast genomes, and that length polymor-phism occurs primarily within these regions. A clustering ofpolymorphic microsatellites in therpl23 region of the ricechloroplast genome was recently reported by Provan et al.(1996). The existence of mutational hotspots in certainnoncoding cpDNA regions has been emphasized in severalstudies, but gene conversion and recombinational processes,rather than replication slippage, were suggested as responsi-ble for the observed insertion and deletion events (Mortonand Clegg 1993; Johnson and Hattori 1996; Goulding et al.1996). An alternative explanation is that variable mono-nucleotide repeats are present anywhere in noncodingcpDNA, and that any intronic and intergenic cpDNA regionwould therefore show high levels of length variation if sepa-rated on sequencing gels. This view is supported by VanHam et al. (1994), who found a total of 50 small insertionand deletion mutations (partly due to mononucleotide repeatvariation) in the intergenictrnL–trnF spacer of 15 speciesbelonging to the families Crassulaceae, Saxifragaceae, andSolanaceae.

Regardless of which mutational and (or) evolutionaryforces are involved, the consensus primers designed in thepresent study clearly provide a high probability of detectingpolymorphic PCR products. It should be stressed that lengthpolymorphisms caused by variable mononucleotide repeatsare certainly not suitable for phylogenetic studies due totheir presumably high mutation rates and the associated riskof homoplasy (which is probably also true for nuclearmicrosatellites, see Ortí et al. 1997). However, the techniquehas several inherent advantages for discriminating closelyrelated genotypes. First, length variants are directly dis-played on the gels, obviating the need to test large numbersof restriction enzymes. Second, haplotype data can be gener-ated by analyzing several loci on a single gel lane. Third,and most importantly, chloroplast haplotypes and nuclearmicrosatellite alleles can be evaluated simultaneously bymultiplex PCR in combination with automated fluorescencesizing. The concurrent analysis of independently inheriteduni- and biparental markers will facilitate studies on the rel-ative contribution of seed and pollen movement to inter-populational gene flow (McCauley 1994; Powell et al.

© 1999 NRC Canada

Weising and Gardner 17

1996b; Petit et al. 1997), introgressive hybridization events(as exemplified byN. glutinosaandN. tabacumin the pres-ent study), the origin of polyploids (Provan et al. 1997), andlast, but not least, maternalvs. paternal chloroplast inheri-tance in disputed cases (Cato and Richardson 1996). RFLPstudies have indicated a strictly paternal mode of cpDNA in-heritance in interspecific crosses within the genusActinidia(Cipriani et al. 1995; Testolin and Cipriani 1997). We arecurrently using ccmp pairs to test whether this exceptionalinheritance pattern does also hold at the intraspecific level.

This work was funded by the New Zealand Foundationfor Research Science and Technology (93–07–249). K.W.was supported by the University of Auckland Foundation.We acknowledge the technical help by R.W. Fung and J.Keeling. The experiments performed in the present studycomply with the current laws of New Zealand.

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