9

A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms
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 in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms. Key words: genetic markers, chloroplast genome, microsatellites, consensus primers, angiosperms. Rsum : De courts sries de rptitions mononuclotidiques sont prsentes dans les gnomes chloroplastiques des plantes suprieures. Chez le soya, le riz et le pin, la PCR ralise laide damorces adjacentes ces microsatellites a montr que le nombre de rsidus A ou T est variable parmi des taxons trs apparents. Ici, les auteurs dcrivent des amorces qui permettent dtudier la variation au niveau des microsatellites de lADN chloroplastique chez les angiospermes, un groupe dorganismes pour lequel linformation prsente dans les bases de donnes est limite. Un total de 39 rptitions (A)n et (T)n (n 10) ont t identifies dans le gnome chloroplastique du tabac et les squences nuclotidiques de ces 39 rgions ont t alignes avec des squences orthologues trouves dans les bases de donnes. Des paires damorces consensuelles ont t prpares et utilises pour amplifier lADN total dun ensemble hirarchique dangiospermes. Les dix paires damorces ont permis dobtenir des produits PCR chez des membres des solanaces et huit des dix paires se sont avres fructueuses chez la plupart des autres angiospermes. Le degr de polymorphisme interspcifique lintrieure des genres Nicotiana, Lycopersicon (tous deux des solanaces), et Actinidia (actinidiaces) sest avr lev tandis que la variation intraspcifique chez le Nicotiana tabacum, le Lycopersicon esculentum et lActinidia chinensis tait limite. Le squenage des produits PCR obtenus laide de trois paires a rvl que le nombre variable de rsidus A, G et T tait la principale cause du polymorphisme chez lActinidia. Ces rsultats suggrent que des amorces universelles permettant damplifier des microsatellites peuvent servir doutils gnraux pour tudier la variation chloroplastique chez les angiospermes. Mots cls : marqueurs gntiques, gnome chloroplastique, microsatellites, amorces consensuelles, angiospermes. [Traduit par la Rdaction] Weising and Gardner 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.
1

19

Author 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).
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The conserved nature of the chloroplast genome in higher plants (Wolfe et al. 1987) has some practical implications for genetic research. Most importantly, the high degree of sequence conservation has facilitated the use of heterologous hybridization probes and polymerase chain reaction (PCR) primers in unrelated species, thereby circumventing the need to clone chloroplast DNA (cpDNA) from each and every species under study (Olmstead and Palmer 1994). Universal PCR primer pairs have been constructed on the basis of conserved coding sequences of cpDNA genes and used to amplify the DNA located between the primer binding sites (Taberlet et al. 1991; Demesure et al. 1995; DumolinLapegue et al. 1997). Direct sequencing or restriction analysis of the PCR products often yielded informative polymorphisms, which were exploited to study taxonomic and phylogenetic relationships at various systematic levels (Arnold et al. 1991; Cipriani et al. 1995; Testolin and Cipriani 1997; Wolfe et al. 1997). An undesirable consequence of the extensive cpDNA sequence conservation is the difficulty to discriminate between closely related chloroplast genomes. While intraspecific cpDNA restriction site variation has been reported for many species (reviewed by Soltis et al. 1992), the magnitude of such variability is generally low. The need to test many restriction enzymes in order to detect a single polymorphism makes the search for intraspecific cpDNA variation quite cumbersome, even when assisted by PCR with universal primers. Recently, a new cpDNA marker system has been established that is based on the occurrence of a certain type of microsatellite in the chloroplast genome (Powell et al. 1995a, 1995b). Microsatellites, also called simple sequence repeats, are abundant polymorphic elements of eukaryotic nuclear genomes and consist of tandemly reiterated, short DNA sequence motifs (Wang et al. 1994; Field and Wills 1996). Size variation of microsatellites is due to a variable repeat copy number and can be visualized by PCR with pairs of flanking primers and electrophoretic separation of the amplification products. Nuclear microsatellites are often multiallelic within and among populations, inherited in a codominant fashion, and fast and easy to type. They have therefore become the marker system of choice for genetic mapping, population genetics, and DNA profiling in plants (Powell et al. 1996a; Weising et al. 1998). Database surveys have shown that microsatellites are comparatively rare in organellar DNA (Wang et al. 1994). In fact, the only type of simple repeat found in the chloroplast genome of higher plants is made up of short poly(A) or poly(T) tracts, with maximum sizes of about 20 bp. Nevertheless, a number of studies employing flanking PCR primers have shown that such mononucleotide stretches are polymorphic among different species and accessions of Glycine (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). Wherever the PCR products were analyzed by DNA sequencing, the length variability was found to be due to a variable number of A or T residues. These observations suggest that simple sequence repeats in chloroplasts may provide a general marker system for evaluating the genetic structure of plant

populations and for studying the mode of chloroplast inheritance. As with nuclear microsatellites, a more widespread use of the approach is currently limited by the need of sequence data for primer construction. In all studies published so far, primer pair sequences for the amplification of chloroplast microsatellites were deduced from database entries. We are interested in applying chloroplast markers for inheritance studies in wild and cultivated kiwifruit (Actinidia deliciosa and relatives), where database information is limited. One way to approach this problem would be the construction of consensus primer pairs designed to amplify chloroplast microsatellites in many different plant species. Such primer pairs would have to meet three criteria. First, their target sequences need to be sufficiently conserved to generate a fragment from unrelated species. Second, the amplification products should be polymorphic, e.g., by harbouring a variable mononucleotide repeat. Third, the products should be sufficiently short to allow single-base resolution on sequencing gels. Here we describe a set of 10 consensus primer pairs based on multiple alignment of mononucleotide repeat-flanking regions in cpDNA from several mono- and dicotyledonous plant species. We show that 8 of the 10 primer pairs are ubiquitously applicable across dicotyledonous angiosperms, and reveal intra- and interspecific cpDNA polymorphisms within the genera Nicotiana, Lycopersicon, and Actinidia.

Plant materials
All Actinidia plant material originated from Hort Research orchards, Kumeu and Te Puke, New Zealand. Tomato species and cultivars were derived from the Botanical Garden, University of Frankfurt, Germany. A set of 12 N. tabacum cultivars was derived from Nelson Research Centre, Nelson, New Zealand. Origins of all other plant material used in the present study are summarized in Table 2. Leaves were either used fresh, or frozen in liquid nitrogen, and stored at 80C. Pinus radiata DNA was a gift from T. Richardson, Forest Research Institute, Rotorua, New Zealand.

DNA isolation
Genomic DNA was isolated from young leaves of different angiosperm species using various modifications of the cetyl trimethylammonium (CTAB) procedure (Weising et al. 1995, 1996). DNA concentrations were determined electrophoretically against known amounts of DNA as standards. For PCR, DNA samples were adjusted to a concentration of 20 ng/ L.

Database studies
The Genetic Computer Group (GCG) software package (Devereux et al. 1984) was used for all DNA sequence analyses except for the homology searches (see below). Initially, the fully sequenced tobacco chloroplast genome (Shinozaki et al. 1986) was screened for the presence of mononucleotide runs with n 10 using FINDPATTERNS. For each candidate, 400 bp of sequence encompassing the repeat were then excised using SEQED, and screened for homologies in the EMBL and GenBank databases using BLASTN (Altschul et al. 1990). Finally, the PILEUP software was applied to generate multiple alignments of homologous sequences with the respective tobacco cpDNA region.
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11 product was checked by agarose electrophoresis, and the remainder was purified through a High Pure PCR Purification Kit (Boehringer Mannheim). Double-stranded sequencing was performed by the dideoxynucleotide chain termination method using an Applied Biosystems 373A DNA sequencer. Both strands were sequenced for each product. Sequences have been deposited in the DDJB nucleotide sequence database (accession numbers AB006089AB006101). The LINEUP and PILEUP programs of the GCG package were used to generate multiple sequence alignments (Devereux et al. 1984).

Primer design
From the alignment information, 10 loci were selected for consensus primer pair construction using the program Cprimer (written by G. Bristol and R.D. Anderson, School of Medicine, University of California, Los Angeles, Cal.) which is available from the Internet (see Results section for the criteria for candidate selection). Primer pairs were checked for the absence of self-annealing, dimer and hairpin formation capacities. Primer spacing was chosen to result in PCR products with an expected size range of 80160 bp in tobacco. Primers fulfilling all criteria were purchased from Gibco-BRL. A set of 20 additional primer pairs (MapPairs) specific for Pinus thunbergii chloroplast microsatellites (Vendramin et al. 1996) was purchased from Research Genetics.

Radioactive PCR was performed in 10 L volumes using a Techne PHC-3 thermal cycler. Each reaction contained 20 ng of template DNA, 2.5 mM MgCl2, 0.5 M each of forward and reverse primer, 0.2 mM each of dATP, dGTP, and dTTP, 0.02 mM of dCTP, 0.04 L of 3000 Ci/mmole [32P]dCTP or [33P]dCTP, 10 mM TrisHCl at pH 8.3, 50 mM KCl, and 1 unit AmpliTaq DNA polymerase (Perkin Elmer). Reactions were overlayed with two drops of mineral oil. The PCR regime generally followed the conditions described by Vendramin et al. (1996), but with a lower annealing temperature. After an initial denaturation at 94C for 5 min, PCR was performed for 30 cycles, each consisting of 94C for 1 min, 50C for 1 min, and 72C for 1 min. Final extension was at 72C for 8 min. PCR products were mixed with 1 vol. of 97.5% (v/v) formamide, 10 mM EDTA at pH 7.5, 0.3% (w/v) xylene cyanol, 0.3% (w/v) bromophenol blue, and were denatured at 95C for 3 min. Aliquots of each sample were electrophoresed on denaturing polyacrylamide gels (6% acrylamidebisacrylamide (19:1), 8 M urea in TrisborateEDTA buffer, pH 8.3) at constant power (55 W) for 23 h. Sequencing reactions of M13mp9 single-stranded DNA (Boehringer Mannheim) were used as molecular weight standards. The gels were dried at 80C for 45 min, and exposed to Kodak XR5 film without intensifying screens for 324 h. Bands were scored by inspection, with the allele sizes calculated from the most intense band.

Microsatellite analysis using radioisotopes

Fluorescent PCR was performed in 10 L volumes using a GeneAmp 2400 thermocycler (Perkin Elmer). Fluorescent dUTPs were purchased from Perkin Elmer. Each reaction contained 20 ng of template DNA, 2.5 mM MgCl2, 0.5 M each of forward and reverse primer, 0.2 mM of each dNTP, 10 mM TrisHCl at pH 8.3, 50 mM KCl, 1 unit AmpliTaq DNA polymerase (Perkin Elmer) and either 1 M dUTP[R6-G] (green), 1 M dUTP[R110] (blue), or 4 M dUTP[TAMRA] (yellow). The same PCR regime was used as described above. PCR products containing each of three different 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% acrylamidebisacrylamide (19:1) and 8 M urea in TBE at pH 8.3) using an Applied Biosystems 373A DNA sequencer. Fragment mobilities were measured by real-time laser scanning, and sizes were determined using the ABIPRISM GeneScan and Genotyper software packages (Perkin Elmer).

Microsatellite analysis using fluorescent dyes

Sequencing of PCR fragments

The PCR products from 16 primer-template combinations were characterized by direct DNA sequencing. Large-scale PCR was performed in 100 L volumes as described above, but omitting fluorescent and radiolabelled nucleotides. An aliquot of the PCR

Design of consensus primer pairs that flank chloroplast microsatellites The initial objective of the present study was to develop primer pairs that detect cpDNA polymorphisms among cultivated kiwifruit (Actinidia deliciosa) and related species. Recently, Cato and Richardson (1996) have reported successful amplification of Actinidia chinensis cpDNA, using two out of five primer pairs derived from mononucleotide repeatflanking regions of Pinus thunbergii, a gymnosperm. Based upon this observation, we tested a set of 20 primer pairs designed for the amplification of pine chloroplast microsatellites (Vendramin et al. 1996) with total DNA from A. chinensis, using Pinus radiata DNA as a positive control. All primer pairs produced bands with Pinus DNA as expected, but only one pair reproducibly yielded a single PCR product in the expected size range with A. chinensis DNA (not shown). All other primers either revealed no product, a smear, or a multilocus pattern when reduced stringency conditions were applied. Analyzing the only functional primer pair on a set of A. chinensis accessions on a sequencing gel showed no size variation. The conclusion from these experiments was that conservation of mononucleotide repeatflanking regions between gymno- and angiosperms is quite poor. We therefore decided to design a set of primer pairs solely based on angiosperm cpDNA. The completely sequenced chloroplast genome of Nicotiana tabacum (Shinozaki et al. 1986) was used as a starting point. Screening the tobacco cpDNA for mononucleotide arrays with n 10 yielded 39 poly(A) and poly(T) repeats, whereas extended poly(G) or poly(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, inverted repeat regions, the total number of useful candidates dropped to 33. EMBL and GenBank databases were screened for cpDNA sequences homologous to each candidate region, and orthologous regions were aligned to 400 bp segments encompassing the tobacco repeats. Reasonable alignments were possible for 25 of the 33 candidates (alignments are available from the authors upon request). Two criteria were applied to select the most promising candidates for primer pair design. First, only those loci were considered where putative primer target regions flanking the mononucleotide repeat were sufficiently conserved. In this respect, particular attention was given to the primer 3 end. Second, the sequence internal to the primer binding sites should harbour a long poly(A) or poly(T) tract not only in tobacco, but also in other species (which was the case in about half of the candidates). We reasoned that the first cri 1999 NRC Canada

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Table 1. Size and position of 10 tobacco cpDNA microsatellites selected for the construction of consensus primer pairs ccmp1ccmp10. Tm values were calculated by the Wallace rule (Thein and Wallace 1986), or according to the Cprimer program (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). Position (bp) 3801 8609 10 075 12 872 16 950 16 977 45 119 57 339 71 563 74 060 86 694 Repeat in tobacco (T)10 (A)11 (T)11 (T)13 (C)7(T)10 (T)5C(A)11 (T)5C(T)17 (A)13 (T)6C(T)14 (T)11 (T)14 Primers deduced from multiple sequence alignment 5-CAGGTAAACTTCTCAACGGA-3 5 -CCGAAGTCAAAAGAGCGATT-3 5-GATCCCGGACGTAATCCTG-3 5-ATCGTACCGAGGGTTCGAAT-3 5-CAGACCAAAAGCTGACATAG-3 5-GTTTCATTCGGCTCCTTTAT-3 5-AATGCTGAATCGAYGACCTA-3 5-CCAAAATATTBGGAGGACTCT-3 5-TGTTCCAATATCTTCTTGTCATTT-3 5-AGGTTCCATCGGAACAATTAT-3 5-CGATGCATATGTAGAAAGCC-3 5-CATTACGTGCGACTATCTCC-3 5-CAACATATACCACTGTCAAG-3 5-ACATCATTATTGTATACTCTTTC-3 5-TTGGCTACTCTAACCTTCCC-3 5-TTCTTTCTTATTTCGCAGDGAA-3 5-GGATTTGTACATATAGGACA-3 5-CTCAACTCTAAGAAATACTTG-3 5-TTTTTTTTTAGTGAACGTGTCA-3 5-TTCGTCGDCGTAGTAAATAG-3 Tm (C) Wal 58 58 60 60 58 56 60 58 62 58 58 60 56 58 60 58 54 56 56 58 Cpr 53.3 57.3 58.0 58.6 51.3 53.9 55.3 56.1 55.0 55.4 52.7 52.5 45.1 44.5 52.9 53.9 45.1 44.2 53.3 53.7 Size in tobacco (bp) 139 189 112 126 121 103 133 77 98 103

Code ccmp1 ccmp2 ccmp3 ccmp4 ccmp5 ccmp6 ccmp7 ccmp8 ccmp9 ccmp10

Location trnK intron 5 to trnS trnG intron atpF intron 3 to rps2 ORF 77ORF 82 intergenic atpBrbcL intergenic rpl20rps12 intergenic ORF 74bpsbB intergenic rpl2rps19 intergenic

terion would help to maximize the transportability of primers across taxa, while the second criterion would increase the chance of size variation. Primer pairs were designed for 10 candidates which met both criteria best. We named this set of primers: consensus chloroplast microsatellite primers (ccmp), ccmp1 to ccmp10. Their sequences, calculated Tm values, as well as the size, type, and location of the corresponding microsatellite repeat in the tobacco cpDNA, are summarized in Table 1. From the sequence alignments, the most promising candidate was ccmp10, amplifying the rpl2rps19 intergenic region, with poly(A) tracts of variable size in almost all species examined (see also Goulding et al. 1996). Screening of consensus chloroplast microsatellite primers with a set of angiosperms All 10 primer pairs were tested with a hierarchically structured DNA template set, consisting of 13 samples from the nightshade family (including the genera Nicotiana, Lycopersicon, Petunia, and Solanum), five Actinidia species, six other dicotyledons from various families, and three monocots (Table 2). Total leaf DNA was amplified in the presence of radioactive dCTP, and the PCR products were separated on sequencing gels and visualized by autoradiography. A standard PCR protocol (with an annealing temperature of 50C) was used for all primer pairs, irrespective of the calculated Tm values (Table 1). The results obtained with two primer pairs are shown in Fig. 1. The allele sizes obtained with all primer pairs across all the species are compiled in Table 2.

The results can be summarized as follows: (i) All primer pairs produced a fragment of the expected size from Nicotiana tabacum. All other Solanacean species were also amplified, with the exception of ccmp8 which failed to produce a product with two templates. Moreover, 8 of the 10 primer pairs (i.e., ccmp17 and ccmp10) also generated products for the majority of tested species from other dicotyledonous angiosperm families, while amplification of monocotyledons was somewhat less efficient. (ii) PCR products showed considerable size variation, not only among the investigated genera and families, but also within genera. Alleles were often separated by steps of 1 bp, which is consistent with a variable number of A or T residues in a mononucleotide repeat. (iii) No intraspecific variation was found in this series of experiments. Also, the Actinidia deliciosa and A. chinensis samples were always identical, which supports the close relationship of these two species (Cipriani and Morgante 1993; Atkinson et al. 1997). With one exception (ccmp2), identical product sizes were also observed between Nicotiana tabacum and N. sylvestris which is considered to be a progenitor of the allotetraploid cultivated tobacco (Gray et al. 1974). The well-known stuttering phenomenon, which usually occurs upon amplification of mono- and dinucleotide-type microsatellites, was observed with all primers. This effect is thought to be a consequence of polymerase slippage during replication (Litt et al. 1993). Comparison of band sizes to the known tobacco sequence showed that the band second to the top is the correct band among the cluster of bands which were separated by one base pair. Differences between
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Table 2. Allele sizes of amplification products from a hierarchical set of angiosperm species, generated by primers ccmp1ccmp10. Radiolabelled DNA fragments were separated on sequencing gels and visualized by autoradiography. An asterisk indicates that more than a single band was amplified by the respective primertemplate combination. In this case, the allele size of the strongest band is given. Size of amplification products (bp) Lane a b c d e f g h i j k l m n o p q r s t u v w x y z Species Nicotiana tabacum cv. Petit Havana (FRA) N. tabacum cv. Virginia (FRA) N. tabacum cv. Atropurpurea (FRA) N. silvestris (FRA) N. acuminata (FRA) N. benthamiana (AUK) Lycopersicon hirsutum (FRA) L. peruvianum (FRA) L. esculentum var. finiens (FRA) L. esculentum cv. Haubners Vollendung (FRA) L. esculentum cv. UC82B (HORT) Petunia hybrida (AUK) Solanum tuberosum (FRA) Actinidia arguta (HORT) A. polygama (HORT) A. chrysantha (HORT) A. chinensis (HORT) A. deliciosa var. coloris (HORT) Brassica oleracea (AUK) Sinapis alba (AUK) Arabidopsis thaliana (AUK) Pisum sativum (AUK) Metrosideros excelsa (AUK) Malus domestica (HORT) Hordeum vulgare (AUK) Lolium multiflorum (AUK) Cordyline australis (AUK) Family Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Actinidiaceae Actinidiaceae Actinidiaceae Actinidiaceae Actinidiaceae Brassicaceae Brassicaceae Brassicaceae Fabaceae Myrtaceae Rosaceae Poaceae Poaceae Agavaceae ccmp1 139 139 139 139 134 139 140 143 141 141 141 138 140 131 132 131 130 130 139 122 140 139 139* 127 139 139 139 ccmp2 189 189 189 188 188 189 189 188 190 190 190 193 188 206 208 210 209 209 166 165 158 234 211 196 182* 186* 187* ccmp3 112 112 112 112 113 109 114 113 114 114 114 108 115 114 114 107 107 107 103* 104 103 93 119 100 89 ccmp4 126 126 126 126 123 128 123 123 122 122 122 125 124 138 137 138 138 138 128* 124 124 115 135 126 ~220 168 ccmp5 121 121 121 121 119 118 119 118 119 119 119 118 119 89 98 98 98 98 119 109 107 122 145 160 77 ccmp6 103 103 103 103 101 100 93 93 93 93 93 96 93 102 102 102 102 102 93 94 96 111 103 106 98 95 100 ccmp7 133 133 133 133 129 130 133 133 134 134 134 131 133 135 133 132 131 131 140 140 140 146 151 132 130 130 130* ccmp8 77 77 77 77 77 65 65 65 65 65 66 ccmp9 99 99 99 99 103 101 101 96 101 101 101 104 98 101 101* 101 101* 101 101 ccmp10 103 103 103 103 102 109 110 111 109 110 110 113 110 91 91 92 91 91 96 96 95 ~230 98 113 96* 96* >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). 1999 NRC Canada

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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). M13mp9 sequencing reactions served as molecular weight markers.

samples were easily recognized, because the whole clusters were 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 intraspecific polymorphisms was due to the limited number of accessions examined, we next analyzed a broader set of samples, including 12 additional tobacco varieties, two more Nicotiana species (N. otophora and N. glutinosa), six additional tomato cultivars, the wild tomato species Lycopersicon pimpinellifolium, and 22 accessions belonging to 10 different Actinidia species (including all samples from Table 2 plus A. guilinensis, A. hemsleyana, A. macrosperma, A. melanandra, and A. setosa). To achieve a higher sample throughput, PCR products from different primer/template

combinations were pooled and analyzed by automated fluorescence detection (Ziegle et al. 1992; Diwan and Cregan 1997). Some samples were evaluated by both fluorescence and radioactivity to test the comparability of both approaches. Fragment sizes automatically called by the Genescan and Genotyper softwares were in a range of 0.6 bp of those detected by autoradiography. This is similar in magnitude to variation found in studies on nuclear microsatellites (Diwan and Cregan 1997). Provided that fluorescence gels were run at high resolution and peaks were evaluated carefully, consistent size differences between fragments were obtained with both techniques. Allele sizes revealed by the fluorescence approach are summarized in Table 3. The full data sets obtained for individual samples are available from the authors upon request. Again, there was considerable interspecific variation within
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15 102, 103, 105, 109, 122 103, 105 90, 91, 92, 93 109, 110, 111 96, 100, 101 133, 134 118, 119, 120 122, 123 113, 114 188, 189, 190 140, 141, 143 93

Table 3. Summary of allele sizes of amplification products generated by primers ccmp1ccmp7, ccmp9, and ccmp10 from a set of 6 Nicotiana species, 15 N. tabacum cultivars, 4 Lycopersicon species, 8 L. esculentum cultivars, 10 Actinidia species, and 10 A. chinensis accessions. Fluorescent-labelled PCR products were separated on sequencing gels and fragment sizes determined by ABIPRISM GeneScan and Genotyper software packages.

Sizes of amplification products (bp)

Nicotiana, with 3 to 5 different band sizes among six species. However, intraspecific variation among N. tabacum cultivars was limited, with two notable exceptions. First, primer pair ccmp2 yielded alleles of 189 and 190 bp, with similar frequency among the individual cultivars of N. tabacum and the diploid N. sylvestris. Second, the chloroplast haplotype of N. tabacum Kentucky 34 was completely identical to that of N. glutinosa (see Discussion). In tomato, most alleles were shared between Lycopersicon esculentum and its putative progenitor, L. pimpinellifolium. Intraspecific polymorphisms were observed with primer pairs ccmp9 and 10 only, both detecting two alleles each (Table 3). Analysis of the Actinidia samples revealed between one and five size variants among the 10 species examined (Table 3). Each species was characterized by a unique haplotype, except for A. deliciosa which was identical to the most common haplotype found in A. 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 situation in tobacco and tomato, the extent of intraspecific polymorphism was limited. Variation was only observed with primer pairs ccmp3 and ccmp10, which detected two alleles each among the eight diploid A. chinensis accessions examined. These allowed discrimination of three haplotypes. The more common allele of each pair was also present in tetraploid A.chinensis and hexaploid A. deliciosa varieties. Sequence analysis of cpDNA amplification products from Actinidia species To credibly examine whether the observed size differences were due to microsatellite variation, or other mutational events, we sequenced the PCR products obtained from 3 primer pairs and 5 Actinidia species, and aligned these sequences to the corresponding tobacco sequence derived from the database (Fig. 2). The alignments demonstrate that variable numbers of mononucleotide repeats are in fact the major cause of polymorphism among Actinidia species. Primer pairs ccmp1, 2, and 7 generated a total of 3, 4, or 5 variants, respectively. The three ccmp1 variants simply differ by the number of Ts (T10 vs. T11 vs. T12), while the other loci are somewhat more complex. In case of ccmp7, runs of both A and G residues were found responsible for the observed variability, reminiscent of the situation in a soybean tRNAMet gene (Powell et al. 1995b). In all three cases, A. chinensis and A. deliciosa alleles were completely identical. Quite unexpectedly, the mononucleotide repeats responsible for the polymorphism among Actinidia species were not necessarily those originally selected for the alignment (underlined in Fig. 2). Instead, variability was found within adjacent shorter repeats in two of three regions sequenced.

ccmp10

98, 99, 101, 103 99, 103

ccmp9

nd

ccmp7

127, 129, 130, 132, 133 132, 133

131, 132, 133, 134, 135 131

ccmp6

96, 100, 101, 103 100, 103

102

102

ccmp5

116, 118, 119, 121, 125 121, 125

89, 98

98

ccmp4

123, 124, 126, 128 124, 126

137, 138

107, 114, 115, 122 107, 122

138

ccmp3

109, 110, 112, 113, 130 112, 130

206, 208, 209, 210, 211 209

188, 189, 190

ccmp2

189,190

130, 131, 132

ccmp1

134, 139, 140

130

139

We have applied a consensus PCR primer concept to the study of small length polymorphisms caused by variable numbers of A and T residues in mononucleotide repeats in cpDNA (Powell et al. 1995a, 1995b). A serious challenge for primer design was that PCR products had to be suffi-

Actinidia (10 species) Actinidia chinensis (10 accessions) Nicotiana (6 species) Nicotiana tabacum (15 cultivars) Lycopersicon (4 species) Lycopersicon esculentum (8 cultivars)
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141

190

114

122

119

93

134

100, 101

nd

109, 110

90,91

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Fig. 2. Alignment of cpDNA sequences from Actinidia species amplified by primer pairs ccmp1, ccmp2, and ccmp7 with the corresponding sequences of the Nicotiana tabacum cpDNA (Shinozaki et al. 1986). The repeat containing the length polymorphism is shown in bold, as are other nucleotide differences between the Actinidia sequences. The long repeat originally targeted in the tobacco alignment is double-underlined. All A. deliciosa sequences were completely identical to the corresponding A. chinensis sequences and were therefore omitted. ccmp 1 1 TTCTCTATCC TTCTCTATCC TTCTCTATCC TTCTCTATCC TTCTCTATCA 51 AGGAGAAGAA AGGAGAAGAA AGGAGAAGAA AGGAGAAGAA AGTGTTATAG 50 TGTTCGTTAT TGTTCGTTAT TGTTCGTTAT TGTTCGTTAT TCTATGTTAT 100 TTGCAACCCA TTGCAACCCA TTGCAACCCA TTGCAACCCA TTTCAACCTA

A. A. A. A. N. A. A. A. A. N.

chinensis chrysantha arguta polygama tabacum chinensis chryeantha arguta polygama tabacum

TCTCTTTTTC TCTCTTTTTC TCTCTTTTTC TCTCTTTTTC TCTCTTTTTT GACGGTTAGA GACGGTTAGA GACGGTTAGA GACGGTTAGA GATAATAAGA

CATTTAATGG CATTTAATGG CATTTAATTG CATTTAAGTG TTTTCGTTTC .AATCCTTTA .AATCCTTTA .AATCCTTTA .AATCCTTTA TGGTTAGAAA

GTTTA..... GTTTA..... GTTTA..... GTTTA..... GTTTAATTGG ..TTTTTTTT .TTTTTTTTT .TTTTTTTTT TTTTTTTTTT TCCTTTATTT

ccmp 2

A. A. A. A. A. N. A. A. A. A. A. N. A. A. A. A. A. N.

chinensis chryeantha setosa arguta polygama tabacum chinensis chrysantha setosa arguta polygama tabacum chinensis chrysantha setosa arguta polygama tabacum

1 AAAATAAAAA AAAATAAAAA AAAATAAAAA AAAATAAAAA AAAATAAAAA AAAATAAAAA 51 CTTAGAGGTT CTTAGAGGTT CTTAGAGGTT CTTAGAGGTT CTTAGAGGTT CTTATGATTT 101 TTTGCAAAAT TTTGCAAAAT TTTGCAAAAT TTTGCAAAAT TTTGCAAAAT TTTCGAAATT

.GGTTTTCGT .GGTTTTCGT .GGTTTTCGT .GGTTTTCGT .GGTTTTCGT AGGT.....T TATATATTTC TATATATTTC TATATATTTC TATATATTTC TATATATTTC GGTCTATTCC TTGAAAGAGA TTGAAAGAGA TTGAAAGAGA TTGAAAGAAA TTGAAAGCGA TGAAAAAAAA

TTTTCTTGCT TTTTCTTGCT TTTTCTTGCT TTTTCTTGCT TTTTCTTGCT TTTCCTTGCT ACACGTTTAA ACACGTTTAA ACACGTTTAA ACACGTTTAA ACACGTTTAA ACACATTTAA AATCAAATAT AATAAAATAT AATCAAATAT AATCAAATAT AATCAAAGAT AA......AT

TGATTT..AA TGATTT.AAA TGATTTAAAA TGATTTT... TGATT...AA TGATTTT... CTACGAAAAA CTACGAAAAA CTACGAAAAA CTACGAAAAA CTACGAAAAA CTAAGAATAA CAAGTCATCC CAAGTCATCC CAAGTCATCC CAAGTCATCC CAAGTCATAC CAAGTCATC.

50 AAAAAAAATT AAAAAAAATT AAAAAAAATT ..AAAAAATT AAAAAAAATT ..CCAATTTT 100 AGAAAAGAGA AGAAAAGAGA AGAAAAGAGA AGAAAAGAGA AGAAAAGAGA GAACAAAGGA 150 AAGGAAACGG AAGGAAACGG AAGGAAACGG AAGGAAACGG AAGGAAACGG .....AACGG

ccmp 7

A. A. A. A. N. A. A. A. A. N.

chinensis polygama chrysantha arguta tabacum chinensis polygama chrysantha arguta tabacum

1 AGGGAATTTC AGGGAATTTC AGGGAATTTC AGGGAATTTC GGGGAAGTTC 51 ..GGGGGGGT A.GGGGGGGT .GGGGGGTGT GGGGGGGGGT AAAAAAAG.T

TTATTCTTT. TTATTCTTT. TTATTCTTT. TTATTCTTTG TTATTATTT. .AAAAATAAG CAAAAATAAG .AAAAATAAG AAAAAATAAG AAAAAAGAAA

AGGTTATTTC AGGTTATTTC AGGTTATTTC AGGTTATTTC AGGTTAGTCA AATTGGGTTG AATTGGGTTG AATTGGGTTG AATGGGGTTG AATTGGGTTG

GGTATTTCGA GGTATTTCGA GGTATTTCGA GGTATTTCGA GGTATTTCCA CGCCATATAT CGCCATATAT CGCCATATAT CGCCATATAT CGCTATATAT

50 TTCAAAAAAA TTCAAAAAAA TTCAAAAAAA TTCAAAAAAA TTTCAAAAAA 92 AT AT AT AT AT

ciently short (<200 bp) to ensure discrimination of one-base differences on sequencing gels. In contrast to previously published universal primer strategies (for example, Taberlet et al. 1991; Demesure et al. 1995; DumolinLapegue et al. 1997), at least one primer of each pair had to

be targeted to noncoding cpDNA. Despite the comparatively low base-substitution rate of cpDNA (Wolfe et al. 1987), non-coding regions in general are not sufficiently conserved to guarantee primer transferability between gymno- and angiosperms (Cato and Richardson 1996, this study). Insufficient
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sequence conservation of non-coding primer target sites proved to be a problem also within angiosperms, and difficulties with alignment reduced the number of promising consensus primer candidate regions from 39 to about 15. Nevertheless, the 10 ccmp pairs that were finally selected worked well within Solanaceae. More importantly, eight of these 10 amplified most other dicotyledonous species, and some primers also worked with the three monotyledons examined. These results demonstrate that the construction of universally applicable consensus primers from mononucleotide-repeat flanking regions in cpDNA is feasible. For monocotyledons, it may be desirable to design an independent consensus primer set based on the completely sequenced rice and maize chloroplast genomes. The analysis of a hierarchical set of species and cultivars showed that PCR products generated by ccmp pairs were polymorphic at various levels. As expected, fragment size variation increased with the phylogenetic distance between the investigated taxa. The extent of variability depended on the locus. The largest size spectrum was observed within the rpl2rps19 intergenic region amplified by ccmp10, ranging from 91 bp for Actinidia up to more than 300 bp for the cabbage tree, Cordyline australis. This same locus has also been found 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 of the particular poly(A) or poly(T) repeat under study. For example, the ccmp6 pair flanked the longest mononucleotide repeat found in the tobacco cpDNA [i.e., (T)5C(T)17], but PCR products generated by this primer pair were among the least polymorphic in all species investigated (Tables 2 and 3). In nuclear microsatellites, the variability (and hence the mutability) of a locus was often shown to be positively correlated with the number of uninterrupted repeats, also in plants (e.g., Saghai-Maroof et al. 1994). The reasons for this contrasting behaviour are not clear, but may relate to different mechanisms of repeat generation and expansion in nuclei versus chloroplasts. Intrageneric variation was considerable in Nicotiana, Lycopersicon, and Actinidia, demonstrating the usefulness of ccmp pairs for detecting polymorphisms below the genus level. However, comparatively few intraspecific polymorphisms were found in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis. Much higher levels of intraspecific chloroplast mononucleotide repeat variation were recently reported from other plant taxa such as soybean (Powell et al. 1995b, 1996b), pine (Powell et al.1995a, Cato and Richardson 1996) and rice (Provan et al.1996, 1997). We consider it unlikely that insufficient sampling caused the paucity of variation observed in the present study, especially in the case of tobacco where a world-wide collection of cultivars was analyzed. It seems more likely that these contrasting results are the consequence of a species-specific component of variation. Restriction site analyses also demonstrated that cpDNA sequence divergence can vary considerably among species (reviewed by Soltis et al. 1992). The low overall level of intraspecific variation among tobacco cultivars made it quite obvious that the chloroplast haplotype of cv. Kentucky 34 was atypical for N. tabacum, but fully compatible with N. glutinosa. This exceptional behaviour can be explained by the breeding history of this

cultivar. About 50 years ago, extensive breeding programs in Kentucky and elsewhere were directed towards the introgression of the N gene (conferring tobacco mosaic virus resistance) from N. glutinosa into cultivated N. tabacum varieties (Valleau 1952). Interspecific crosses between both species were preferably made in one direction, where N. tabacum served as the pollen and N. glutinosa as the seed parent (Valleau 1952). Maternal inheritance of cpDNA, as is assumed to occur in the genus Nicotiana (Scowcroft 1979) would explain the persistence of the N. glutinosa cpDNA haplotype in Kentucky cultivars. Sequencing of amplification products from 16 allele/species combinations showed that variable copy numbers of mononucleotide repeats were a main factor underlying PCR fragment length variation in Actinidia species. At two out of three loci, however, the variability in repeat length was not found in the long mononucleotide stretch selected from the tobacco cpDNA, but occurred in closely adjacent, shorter repeats. There are at least two possible explanations for these surprising results. One is that poly(A) or poly(T) repeats are clustered in chloroplast genomes, and that length polymorphism occurs primarily within these regions. A clustering of polymorphic microsatellites in the rpl23 region of the rice chloroplast genome was recently reported by Provan et al. (1996). The existence of mutational hotspots in certain noncoding cpDNA regions has been emphasized in several studies, but gene conversion and recombinational processes, rather than replication slippage, were suggested as responsible for the observed insertion and deletion events (Morton and Clegg 1993; Johnson and Hattori 1996; Goulding et al. 1996). An alternative explanation is that variable mononucleotide repeats are present anywhere in noncoding cpDNA, and that any intronic and intergenic cpDNA region would therefore show high levels of length variation if separated on sequencing gels. This view is supported by Van Ham et al. (1994), who found a total of 50 small insertion and deletion mutations (partly due to mononucleotide repeat variation) in the intergenic trnLtrnF spacer of 15 species belonging to the families Crassulaceae, Saxifragaceae, and Solanaceae. Regardless of which mutational and (or) evolutionary forces are involved, the consensus primers designed in the present study clearly provide a high probability of detecting polymorphic PCR products. It should be stressed that length polymorphisms caused by variable mononucleotide repeats are certainly not suitable for phylogenetic studies due to their presumably high mutation rates and the associated risk of homoplasy (which is probably also true for nuclear microsatellites, see Ort et al. 1997). However, the technique has several inherent advantages for discriminating closely related genotypes. First, length variants are directly displayed on the gels, obviating the need to test large numbers of restriction enzymes. Second, haplotype data can be generated by analyzing several loci on a single gel lane. Third, and most importantly, chloroplast haplotypes and nuclear microsatellite alleles can be evaluated simultaneously by multiplex PCR in combination with automated fluorescence sizing. The concurrent analysis of independently inherited uni- and biparental markers will facilitate studies on the relative contribution of seed and pollen movement to interpopulational gene flow (McCauley 1994; Powell et al.
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Genome Vol. 42, 1999 Gray, J.C., King, S.D., Wildham, S.G., and Sheen, S.J. 1974. Origin of Nicotiana tabacum L. detected by polypeptide composition of fraction I protein. Nature (London), 252: 226227. Johnson, D.A., and Hattori, J. 1996. Analysis of a hotspot for deletion formation within the intron of the chloroplast trnI gene. Genome, 39: 9991005. Litt, M., Hauge, X., and Sharma, V. 1993. Shadow bands seen when typing polymorphic dinucleotide repeats: Some causes and cures. BioTechniques, 15: 280284. McCauley, D.E. 1994. Contrasting the distribution of chloroplast DNA and allozyme polymorphisms among local populations of Silene alba: Implications for studies of gene flow in plants. Proc. Natl. Acad. Sci. U.S.A. 91: 81278131. Morton, B.R., and Clegg, M.T. 1993. A chloroplast DNA mutational hotspot and gene conversion in a noncoding region near rbcL in the grass family (Poaceae). Curr. Genet. 24: 357365. Olmstead, R.G., and Palmer, J.D. 1994. Chloroplast DNA systematics: A review of methods and data analysis. Am. J. Bot. 81: 12051224. Ort, G., Pearse, D.E., and Avise, J.C. 1997. Phylogenetic assessment of length variation at a microsatellite locus. Proc. Natl. Acad. Sci. U.S.A. 94: 10 745 10 749. Petit, R.J., Pineau, E., Demesure, B., Bacilieri, R., Ducousso, A., and Kremer, A. 1997. Chloroplast DNA footprints of postglacial recolonization by oaks. Proc. Natl. Acad. Sci. U.S.A. 94: 9996 10 001. Powell, W., Morgante, M., McDevitt, R., Vendramin, G.G., and Rafalski, J.A. 1995a. Polymorphic simple sequence repeat regions in chloroplast genomes: Applications to the population genetics of pines. Proc. Natl. Acad. Sci. U.S.A. 92: 77597763. Powell,W., Morgante, M., Andre, C., McNicol. J.W., Machray, G.C., Doyle, J.J., Tingey, S.V., and Rafalski, J.A. 1995b. Hypervariable microsatellites provide a general source of polymorphic DNA markers for the chloroplast genome. Curr. Biol. 5: 10231029. Powell, W., Machray, G.C., and Provan, J. 1996a. Polymorphism revealed by simple sequence repeats. Trends Plant Sci. 1: 215222. Powell, W., Morgante, M., Doyle, J.J., McNicol, J.W., Tingey, S.V., and Rafalski, A.J. 1996b. Genepool variation in genus Glycine subgenus Soja revealed by polymorphic nuclear and chloroplast microsatellites. Genetics, 144: 793803. Provan, J., Corbett, G., Waugh, R., McNicol, J.W., Morgante, M., and Powell, W. 1996. DNA fingerprints of rice (Oryza sativa) obtained from hypervariable chloroplast simple sequence repeats. Proc. Roy. Soc. London B, 263: 12751281. Provan, J., Corbett, G., McNicol, J.W., and Powell, W. 1997. Chloroplast DNA variability in wild and cultivated rice (Oryza spp) revealed by polymorphic chloroplast simple sequence repeats. Genome, 40: 104110. Saghai-Maroof, M.A., Biyashev, R.M., Yang, G.P., Zhang. Q., and Allard, R.W. 1994. Extraordinarily polymorphic microsatellite DNA in barley: Species diversity, chromosomal locations, and population dynamics. Proc. Natl. Acad. Sci. U.S.A. 91: 54665470. Scowcroft, W.R. 1979. Nucleotide polymorphism in chloroplast DNA of Nicotiana debneyi. Theor. Appl. Genet. 55: 133137. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B.Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. 1986. The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO J. 5: 20432049.
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1996b; Petit et al. 1997), introgressive hybridization events (as exemplified by N. glutinosa and N. tabacum in the present study), the origin of polyploids (Provan et al. 1997), and last, but not least, maternal vs. paternal chloroplast inheritance in disputed cases (Cato and Richardson 1996). RFLP studies have indicated a strictly paternal mode of cpDNA inheritance in interspecific crosses within the genus Actinidia (Cipriani et al. 1995; Testolin and Cipriani 1997). We are currently using ccmp pairs to test whether this exceptional inheritance pattern does also hold at the intraspecific level.

This work was funded by the New Zealand Foundation for Research Science and Technology (9307249). 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 study comply with the current laws of New Zealand.

Altschul, S.F., Gish, W., Miller, W., Myers. E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403410. Arnold, M.L., Buckner, C.M, Robinson, J.J. 1991. Pollen-mediated introgression and hybrid speciation in Louisiana irises. Proc. Natl. Acad. Sci. U.S.A. 88: 13981402. Atkinson, R.G, Murray, B., and Gardner, R.C. 1997. The allopolyploid origin of kiwifruit, Actinidia deliciosa (Actinidiaceae). Plant Syst. Evol. 205: 111124. Breslauer, K.J., Frank, R., Blcker, H., and Marky, L.A. 1986. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83: 37463750. Cato, S.A, and Richardson, T.E. 1996. Inter- and intraspecific polymorphism at chloroplast SSR loci and the inheritance of plastids in Pinus radiata D. Don. Theor. Appl. Genet. 93: 587592. Cipriani,G., and Morgante, M. 1993. Evidence of chloroplast DNA variation in the genus Actinidia revealed by restriction analysis of PCR-amplified fragments. J. Genet. Breed. 47: 319326. Cipriani, G., Testolin, R., and Morgante, M. 1995. Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments. Mol. Gen. Genet. 247: 693697. Demesure, B., Sozi, N., and Petit, R.J. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 4: 129131. Devereux, J., Haeberli, P., and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387395. Diwan, N., and Cregan, P.B. 1997. Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean. Theor. Appl. Genet. 95: 723733. Dumolin-Lapegue, S., Pemonge, M.-H., and Petit, R.J. 1997. An enlarged set of consensus primers for the study of organelle DNA in plants. Mol. Ecol. 6: 393397. Field, D., and Wills, C. 1996. Long, polymorphic microsatellites in simple organisms. Proc. Roy. Soc. London B, 263: 209215. Goulding, S.E., Olmstead, R.G., Morden, C.W., and Wolfe, K.H. 1996. Ebb and flow of the chloroplast inverted repeat. Mol. Gen. Genet. 252: 195206.

Weising and Gardner Soltis, D.E., Soltis, P.S., and Milligan, B.G. 1992. Intraspecific chloroplast DNA variation: Systematic and phylogenetic implications. In Molecular systematics of plants. Edited by P.S. Soltis, D.E. Soltis, and J.J. Doyle. Chapman and Hall, New York. pp. 117150. Taberlet, P., Gielly, L., Pautou, G., and Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 17: 11051109. Testolin, R., and Cipriani, G. 1997. Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in the genus Actinidia. Theor. Appl. Genet. 94: 897903. Thein, S.L, and Wallace, R.B. 1986. The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In Human genetic diseases a practical approach. Edited by K.E. Davies. IRL Press, Oxford. p. 3350. Valleau, W.D. 1952. Breeding tobacco for disease resistance. Econ. Bot. 6: 69102. Van Ham, R.C.H.J., t Hart, H., Mes, T.H.M., and Sandbrink, J.M. 1994. Molecular evolution of noncoding regions of the chloroplast genome in the Crassulaceae and related species. Curr. Genet. 25: 558566. Vendramin, G.G., Lelli, L., Rossi, P., and Morgante, M. 1996. A set of primers for the amplification of 20 chloroplast microsatellites in Pinaceae. Mol. Ecol. 5: 595598.

19 Wang, Z., Weber, J.L., Zhong, G., and Tanksley, S.D. 1994. Survey of plant short tandem DNA repeats. Theor. Appl. Genet. 88: 16. Weising, K., Nybom, H., Wolff, K., and Meyer, W. 1995. DNA fingerprinting in plants and fungi. CRC Press, Boca Raton, Flor. Weising, K., Fung, R.W.M., Keeling. J., Atkinson, R.G., and Gardner, R.C. 1996. Characterization of microsatellite repeats from Actinidia chinensis. Mol. Breed. 2: 117131. Weising, K., Winter, P., Httel, B., and Kahl, G. 1998. Microsatellite markers for molecular breeding. J. Crop Production, 1: 113143. Wolfe, K.H., Li, H.-H., and Sharp, P.M. 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast and nuclear DNAs. Proc. Natl. Acad. Sci. U.S.A. 84: 90549058. Wolfe, A.D., Elisens, W.J., Watson, L.E., and DePamphilis, C.W. 1997. Using restriction-site variation of PCR-amplified cpDNA genes for phylogenetic analysis of tribe Cheloneae (Scrophulariaceae). Am. J. Bot. 84: 555564. Ziegle, J.S., Su, Y., Corcoran, K.P., Nie, L., Mayrand, P.E., Hoff, L.B., McBride, L.J., Kronick, M.N., and Diehl, S.R. 1992. Application of automated DNA sizing technology for genotyping microsatellite loci. Genomics, 14: 10261031.\

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