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 ⱖ 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.

Résumé : De courts séries de répétitions mononucléotidiques sont présentes dans les génomes chloroplastiques des
plantes supérieures. Chez le soya, le riz et le pin, la PCR réalisée à l’aide d’amorces adjacentes à ces microsatellites a
montré que le nombre de résidus A ou T est variable parmi des taxons très apparentés. Ici, les auteurs décrivent des
amorces qui permettent d’étudier la variation au niveau des microsatellites de l’ADN chloroplastique chez les
angiospermes, un groupe d’organismes pour lequel l’information présente dans les bases de données est limitée. Un
total de 39 répétitions (A)n et (T)n (n ⱖ 10) ont été identifiées dans le génome chloroplastique du tabac et les
séquences nucléotidiques de ces 39 régions ont été alignées avec des séquences orthologues trouvées dans les bases de
données. Des paires d’amorces consensuelles ont été préparées et utilisées pour amplifier l’ADN total d’un ensemble
hiérarchique d’angiospermes. Les dix paires d’amorces ont permis d’obtenir des produits PCR chez des membres des
solanacées et huit des dix paires se sont avérées fructueuses chez la plupart des autres angiospermes. Le degré de
polymorphisme interspécifique à l’intérieure des genres Nicotiana, Lycopersicon (tous deux des solanacées), et
Actinidia (actinidiacées) s’est avéré élevé tandis que la variation intraspécifique chez le Nicotiana tabacum, le
Lycopersicon esculentum et l’Actinidia chinensis était limitée. Le séquençage des produits PCR obtenus à l’aide de
trois paires a révélé que le nombre variable de résidus A, G et T était la principale cause du polymorphisme chez
l’Actinidia. Ces résultats suggèrent que des amorces universelles permettant d’amplifier des microsatellites peuvent
servir 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

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
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).

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

10
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 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 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 se-
quence 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 re-
striction 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 am-
plification 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. Never-
theless, a number of studies employing flanking PCR prim-
ers 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). 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 general
marker system for evaluating the genetic structure of plant
Genome Vol. 42, 1999
populations and for studying the mode of chloroplast inheri-
tance.
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 se-
quences need to be sufficiently conserved to generate a frag-
ment from unrelated species. Second, the amplification
products should be polymorphic, e.g., by harbouring a vari-
able mononucleotide repeat. Third, the products should be
sufficiently short to allow single-base resolution on sequenc-
ing gels.
Here we describe a set of 10 consensus primer pairs based
on multiple alignment of mononucleotide repeat-flanking re-
gions 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 or-
chards, 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 ni-
trogen, and stored at –80°C. 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 λ DNA as standards. For PCR, DNA
samples were adjusted to a concentration of 20 ng/µL.
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 ⱖ 10 us-
ing FINDPATTERNS. For each candidate, 400 bp of sequence en-
compassing the repeat were then excised using SEQED, and
screened for homologies in the EMBL and GenBank databases us-
ing BLASTN (Altschul et al. 1990). Finally, the PILEUP software
was applied to generate multiple alignments of homologous se-
quences with the respective tobacco cpDNA region.
© 1999 NRC Canada
Weising and Gardner
Primer design
From 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 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 80–160 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.
Microsatellite analysis using radioisotopes
Radioactive PCR was performed in 10 µL volumes using a
Techne PHC-3 thermal cycler. Each reaction contained 20 ng of
template 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 of
dCTP, 0.04 µL 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 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 94°C for
5 min, PCR was performed for 30 cycles, each consisting of 94°C
for 1 min, 50°C for 1 min, and 72°C for 1 min. Final extension
was at 72°C 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 95°C for 3 min. Aliquots of each sample were electrophoresed
on denaturing polyacrylamide gels (6% acrylamide–bisacrylamide
(19:1), 8 M urea in Tris–borate–EDTA buffer, pH 8.3) at constant
power (55 W) for 2–3 h. Sequencing reactions of M13mp9 sin-
gle-stranded DNA (Boehringer Mannheim) were used as molecular
weight 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 calculated
from the most intense band.
Microsatellite analysis using fluorescent dyes
Fluorescent PCR was performed in 10 µL 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 µM 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 1 µM dUTP[R6-G] (green), 1 µM dUTP[R110] (blue),
or 4 µM 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%
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 were
determined using the ABIPRISM GeneScan and Genotyper soft-
ware packages (Perkin Elmer).
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 µL volumes as described above, but omitting flu-
orescent and radiolabelled nucleotides. An aliquot of the PCR
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 per-
formed 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
AB006089–AB006101). The LINEUP and PILEUP programs of
the GCG package were used to generate multiple sequence align-
ments (Devereux et al. 1984).
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 culti-
vated kiwifruit (Actinidia deliciosa) and related species. Re-
cently, Cato and Richardson (1996) have reported successful
amplification of Actinidia chinensis cpDNA, using two out
of five primer pairs derived from mononucleotide repeat-
flanking regions of Pinus thunbergii, a gymnosperm. Based
upon 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 from
A. chinensis, using Pinus radiata DNA as a positive control.
All primer pairs produced bands with Pinus DNA as ex-
pected, 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 con-
ditions 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 experi-
ments was that conservation of mononucleotide repeat-
flanking 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 ⱖ 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 candi-
date region, and orthologous regions were aligned to 400 bp
segments encompassing the tobacco repeats. Reasonable
alignments 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 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
12 Genome Vol. 42, 1999

Table 1. Size and position of 10 tobacco cpDNA microsatellites selected for the construction of consensus primer
pairs ccmp1–ccmp10. 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 Repeat in Primers deduced from multiple Tm (°C) Size in

Code (bp) Location tobacco sequence alignment Wal Cpr tobacco (bp)
ccmp1 3801 trnK intron (T)10 5’-CAGGTAAACTTCTCAACGGA-3’ 58 53.3 139
5’ -CCGAAGTCAAAAGAGCGATT-3’ 58 57.3
ccmp2 8609 5’ to trnS (A)11 5’-GATCCCGGACGTAATCCTG-3’ 60 58.0 189
5’-ATCGTACCGAGGGTTCGAAT-3’ 60 58.6
ccmp3 10 075 trnG intron (T)11 5’-CAGACCAAAAGCTGACATAG-3’ 58 51.3 112
5’-GTTTCATTCGGCTCCTTTAT-3’ 56 53.9
ccmp4 12 872 atpF intron (T)13 5’-AATGCTGAATCGAYGACCTA-3’ 60 55.3 126
5’-CCAAAATATTBGGAGGACTCT-3’ 58 56.1
ccmp5 16 950 3’ to rps2 (C)7(T)10 5’-TGTTCCAATATCTTCTTGTCATTT-3’ 62 55.0 121
16 977 (T)5C(A)11 5’-AGGTTCCATCGGAACAATTAT-3’ 58 55.4
ccmp6 45 119 ORF 77–ORF 82 (T)5C(T)17 5’-CGATGCATATGTAGAAAGCC-3’ 58 52.7 103
intergenic 5’-CATTACGTGCGACTATCTCC-3’ 60 52.5
ccmp7 57 339 atpB–rbcL (A)13 5’-CAACATATACCACTGTCAAG-3’ 56 45.1 133
intergenic 5’-ACATCATTATTGTATACTCTTTC-3’ 58 44.5
ccmp8 71 563 rpl20–rps12 (T)6C(T)14 5’-TTGGCTACTCTAACCTTCCC-3’ 60 52.9 77
intergenic 5’-TTCTTTCTTATTTCGCAGDGAA-3’ 58 53.9
ccmp9 74 060 ORF 74b–psbB (T)11 5’-GGATTTGTACATATAGGACA-3’ 54 45.1 98
intergenic 5’-CTCAACTCTAAGAAATACTTG-3’ 56 44.2
ccmp10 86 694 rpl2–rps19 (T)14 5’-TTTTTTTTTAGTGAACGTGTCA-3’ 56 53.3 103
intergenic 5’-TTCGTCGDCGTAGTAAATAG-3’ 58 53.7

terion would help to maximize the transportability of prim-
ers 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 corre-
sponding microsatellite repeat in the tobacco cpDNA, are
summarized in Table 1. From the sequence alignments, the
most promising candidate was ccmp10, amplifying the
rpl2–rps19 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 struc-
tured 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 auto-
radiography. A standard PCR protocol (with an annealing
temperature of 50°C) was used for all primer pairs, irrespec-
tive of the calculated Tm values (Table 1). The results ob-
tained 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 pro-
duce a product with two templates. Moreover, 8 of the 10
primer pairs (i.e., ccmp1–7 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 prod-
ucts 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 con-
sistent with a variable number of A or T residues in a mono-
nucleotide repeat. (iii) No intraspecific variation was found
in this series of experiments. Also, the Actinidia deliciosa
and A. chinensis samples were always identical, which sup-
ports the close relationship of these two species (Cipriani
and Morgante 1993; Atkinson et al. 1997). With one excep-
tion (ccmp2), identical product sizes were also observed
between Nicotiana tabacum and N. sylvestris which is con-
sidered to be a progenitor of the allotetraploid cultivated to-
bacco (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

© 1999 NRC Canada

 Weising and Gardner
Table 2. Allele sizes of amplification products from a hierarchical set of angiosperm species, generated by primers ccmp1–ccmp10. 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 primer–template
combination. In this case, the allele size of the strongest band is given.
Size of amplification products (bp)
Lane Species Family ccmp1 ccmp2 ccmp3 ccmp4 ccmp5 ccmp6 ccmp7 ccmp8 ccmp9 ccmp10
a Nicotiana tabacum cv. Petit Havana (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103
b N. tabacum cv. Virginia (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103
c N. tabacum cv. Atropurpurea (FRA) Solanaceae 139 189 112 126 121 103 133 77 99 103
d N. silvestris (FRA) Solanaceae 139 188 112 126 121 103 133 77 99 103
e N. acuminata (FRA) Solanaceae 134 188 113 123 119 101 129 — 103 102
f N. benthamiana (AUK) Solanaceae 139 189 109 128 118 100 130 77 101 109
g Lycopersicon hirsutum (FRA) Solanaceae 140 189 114 123 119 93 133 65 101 110
h L. peruvianum (FRA) Solanaceae 143 188 113 123 118 93 133 65 96 111
i L. esculentum var. finiens (FRA) Solanaceae 141 190 114 122 119 93 134 65 101 109
j L. esculentum cv. Haubner’s Vollendung (FRA) Solanaceae 141 190 114 122 119 93 134 65 101 110
k L. esculentum cv. UC82B (HORT) Solanaceae 141 190 114 122 119 93 134 65 101 110
l Petunia hybrida (AUK) Solanaceae 138 193 108 125 118 96 131 — 104 113
m Solanum tuberosum (FRA) Solanaceae 140 188 115 124 119 93 133 66 98 110
n Actinidia arguta (HORT) Actinidiaceae 131 206 114 138 89 102 135 — — 91
o A. polygama (HORT) Actinidiaceae 132 208 114 137 98 102 133 — — 91
p A. chrysantha (HORT) Actinidiaceae 131 210 107 138 98 102 132 — — 92
q A. chinensis (HORT) Actinidiaceae 130 209 107 138 98 102 131 — — 91
r A. deliciosa var. coloris (HORT) Actinidiaceae 130 209 107 138 98 102 131 — — 91
s Brassica oleracea (AUK) Brassicaceae 139 166 103* 128* 119 93 140 — 101 96
t Sinapis alba (AUK) Brassicaceae 122 165 104 124 — 94 140 — — 96
u Arabidopsis thaliana (AUK) Brassicaceae 140 158 103 124 — 96 140 — — 95
v Pisum sativum (AUK) Fabaceae 139 234 93 115 109 111 146 — 101* ~230
w Metrosideros excelsa (AUK) Myrtaceae 139* 211 119 135 107 103 151 — 101 98
x Malus domestica (HORT) Rosaceae 127 196 100 126 122 106 132 — — 113
y 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).
© 1999 NRC Canada

13
14
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, in-
cluding 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 differ-
ent 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
Genome Vol. 42, 1999
combinations were pooled and analyzed by automated fluo-
rescence detection (Ziegle et al. 1992; Diwan and Cregan
1997). Some samples were evaluated by both fluorescence
and radioactivity to test the comparability of both ap-
proaches. 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 flu-
orescence gels were run at high resolution and peaks were
evaluated carefully, consistent size differences between frag-
ments were obtained with both techniques.
Allele sizes revealed by the fluorescence approach are
summarized in Table 3. The full data sets obtained for indi-
vidual samples are available from the authors upon request.
Again, there was considerable interspecific variation within
© 1999 NRC Canada
Weising and Gardner 15

102, 103, 105,

Nicotiana, with 3 to 5 different band sizes among six spe-

90, 91, 92, 93

109, 110, 111

cies. However, intraspecific variation among N. tabacum

109, 122
103, 105

109, 110
cultivars was limited, with two notable exceptions. First,

ccmp10

90,91
primer pair ccmp2 yielded alleles of 189 and 190 bp, with

Table 3. Summary of allele sizes of amplification products generated by primers ccmp1–ccmp7, 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
similar frequency among the individual cultivars of
N. tabacum and the diploid N. sylvestris. Second, the

96, 100, 101

chloroplast haplotype of N. tabacum Kentucky 34 was com-

98, 99, 101,

pletely identical to that of N. glutinosa (see Discussion). In

100, 101
99, 103
ccmp9
tomato, most alleles were shared between Lycopersicon

103
esculentum and its putative progenitor, L. pimpinellifolium.

nd

nd
Intraspecific polymorphisms were observed with primer
pairs ccmp9 and 10 only, both detecting two alleles each

131, 132, 133,

127, 129, 130,

(Table 3).

134, 135

132, 133
Analysis of the Actinidia samples revealed between one

132, 133

133, 134
ccmp7
and five size variants among the 10 species examined

131

134
(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).

101, 103
100, 103
Alleles of different species often differed by steps of 1 bp

96, 100,
ccmp6
(see Table 2), but larger differences were also observed (e.g.,

Sizes of amplification products (bp)

102

102
107, 114, 115, and 122 with ccmp3). Similar to the situation

93

93
in tobacco and tomato, the extent of intraspecific polymor-
phism was limited. Variation was only observed with primer

116, 118, 119,

118, 119, 120

pairs ccmp3 and ccmp10, which detected two alleles each

121, 125
sequencing gels and fragment sizes determined by ABIPRISM GeneScan and Genotyper software packages.
among the eight diploid A. chinensis accessions examined.

121, 125
ccmp5
89, 98
These allowed discrimination of three haplotypes. The more

119
common allele of each pair was also present in tetraploid

98
A.chinensis and hexaploid A. deliciosa varieties.

126, 128
123, 124,
Sequence analysis of cpDNA amplification products
137, 138

124, 126

122, 123
ccmp4
from Actinidia species

138

122
To credibly examine whether the observed size differ-
ences were due to microsatellite variation, or other
mutational events, we sequenced the PCR products obtained
107, 114, 115,

109, 110, 112,

from 3 primer pairs and 5 Actinidia species, and aligned
113, 130
these sequences to the corresponding tobacco sequence de-
107, 122

112, 130

113, 114
rived from the database (Fig. 2). The alignments demon-
ccmp3

122

strate that variable numbers of mononucleotide repeats are

114
in fact the major cause of polymorphism among Actinidia
species. Primer pairs ccmp1, 2, and 7 generated a total of 3,
206, 208, 209,

188, 189, 190

188, 189, 190

4, or 5 variants, respectively. The three ccmp1 variants sim-
210, 211

ply differ by the number of T’s (T10 vs. T11 vs. T12), while
189,190

the other loci are somewhat more complex. In case

ccmp2

of ccmp7, runs of both A and G residues were found respon-

209

190

sible 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 com-
130, 131, 132

134, 139, 140

140, 141, 143

pletely identical. Quite unexpectedly, the mononucleotide re-

peats responsible for the polymorphism among Actinidia
ccmp1

species were not necessarily those originally selected for the

130

139

141

alignment (underlined in Fig. 2). Instead, variability was

found within adjacent shorter repeats in two of three regions
sequenced.
Lycopersicon esculentum
Genus and (or) species

Actinidia chinensis

Nicotiana tabacum
(10 accessions)

(15 cultivars)

(8 cultivars)
(10 species)

We have applied a consensus PCR primer concept to the

(6 species)

(4 species)
Lycopersicon

study of small length polymorphisms caused by variable

Nicotiana
Actinidia

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-

© 1999 NRC Canada

16 Genome Vol. 42, 1999

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 50
A. chinensis TTCTCTATCC TCTCTTTTTC CATTTAATGG GTTTA..... TGTTCGTTAT
A. chrysantha TTCTCTATCC TCTCTTTTTC CATTTAATGG GTTTA..... TGTTCGTTAT
A. arguta TTCTCTATCC TCTCTTTTTC CATTTAATTG GTTTA..... TGTTCGTTAT
A. polygama TTCTCTATCC TCTCTTTTTC CATTTAAGTG GTTTA..... TGTTCGTTAT
N. tabacum TTCTCTATCA TCTCTTTTTT TTTTCGTTTC GTTTAATTGG TCTATGTTAT
51 100
A. chinensis AGGAGAAGAA GACGGTTAGA .AATCCTTTA ..TTTTTTTT TTGCAACCCA
A. chryeantha AGGAGAAGAA GACGGTTAGA .AATCCTTTA .TTTTTTTTT TTGCAACCCA
A. arguta AGGAGAAGAA GACGGTTAGA .AATCCTTTA .TTTTTTTTT TTGCAACCCA
A. polygama AGGAGAAGAA GACGGTTAGA .AATCCTTTA TTTTTTTTTT TTGCAACCCA
N. tabacum AGTGTTATAG GATAATAAGA TGGTTAGAAA TCCTTTATTT TTTCAACCTA

ccmp 2
1 50
A. chinensis AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTT..AA AAAAAAAATT
A. chryeantha AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTT.AAA AAAAAAAATT
A. setosa AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTTAAAA AAAAAAAATT
A. arguta AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATTTT... ..AAAAAATT
A. polygama AAAATAAAAA .GGTTTTCGT TTTTCTTGCT TGATT...AA AAAAAAAATT
N. tabacum AAAATAAAAA AGGT.....T TTTCCTTGCT TGATTTT... ..CCAATTTT
51 100
A. chinensis CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGA
A. chrysantha CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGA
A. setosa CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGA
A. arguta CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGA
A. polygama CTTAGAGGTT TATATATTTC ACACGTTTAA CTACGAAAAA AGAAAAGAGA
N. tabacum CTTATGATTT GGTCTATTCC ACACATTTAA CTAAGAATAA GAACAAAGGA
101 150
A. chinensis TTTGCAAAAT TTGAAAGAGA AATCAAATAT CAAGTCATCC AAGGAAACGG
A. chrysantha TTTGCAAAAT TTGAAAGAGA AATAAAATAT CAAGTCATCC AAGGAAACGG
A. setosa TTTGCAAAAT TTGAAAGAGA AATCAAATAT CAAGTCATCC AAGGAAACGG
A. arguta TTTGCAAAAT TTGAAAGAAA AATCAAATAT CAAGTCATCC AAGGAAACGG
A. polygama TTTGCAAAAT TTGAAAGCGA AATCAAAGAT CAAGTCATAC AAGGAAACGG
N. tabacum TTTCGAAATT TGAAAAAAAA AA......AT CAAGTCATC. .....AACGG

ccmp 7
1 50
A. chinensis AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAA
A. polygama AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAA
A. chrysantha AGGGAATTTC TTATTCTTT. AGGTTATTTC GGTATTTCGA TTCAAAAAAA
A. arguta AGGGAATTTC TTATTCTTTG AGGTTATTTC GGTATTTCGA TTCAAAAAAA
N. tabacum GGGGAAGTTC TTATTATTT. AGGTTAGTCA GGTATTTCCA TTTCAAAAAA
51 92
A. chinensis ..GGGGGGGT .AAAAATAAG AATTGGGTTG CGCCATATAT AT
A. polygama A.GGGGGGGT CAAAAATAAG AATTGGGTTG CGCCATATAT AT
A. chrysantha .GGGGGGTGT .AAAAATAAG AATTGGGTTG CGCCATATAT AT
A. arguta GGGGGGGGGT AAAAAATAAG AATGGGGTTG CGCCATATAT AT
N. tabacum AAAAAAAG.T AAAAAAGAAA AATTGGGTTG CGCTATATAT 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; Dumolin-
Lapegue 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 angio-
sperms (Cato and Richardson 1996, this study). Insufficient

© 1999 NRC Canada

Weising and Gardner
sequence conservation of non-coding primer target sites
proved to be a problem also within angiosperms, and diffi-
culties 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 ex-
amined. These results demonstrate that the construction of
universally applicable consensus primers from mono-
nucleotide-repeat flanking regions in cpDNA is feasible. For
monocotyledons, it may be desirable to design an independ-
ent 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
rpl2–rps19 intergenic region amplified by ccmp10, ranging
from 91 bp for Actinidia up to more than 300 bp for the cab-
bage 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 ex-
ample, 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 cor-
related 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 differ-
ent 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 poly-
morphisms 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 con-
trasting results are the consequence of a species-specific
component 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 chloroplast
haplotype of cv. Kentucky 34 was atypical for N. tabacum,
but fully compatible with N. glutinosa. This exceptional be-
haviour can be explained by the breeding history of this
17
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 va-
rieties (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/spe-
cies 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 re-
peats. 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 polymor-
phism 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 responsi-
ble 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 mono-
nucleotide repeats are present anywhere in noncoding
cpDNA, and that any intronic and intergenic cpDNA region
would therefore show high levels of length variation if sepa-
rated 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 trnL–trnF 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 Ortí et al. 1997). However, the technique
has several inherent advantages for discriminating closely
related genotypes. First, length variants are directly dis-
played on the gels, obviating the need to test large numbers
of 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 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 rel-
ative contribution of seed and pollen movement to inter-
populational gene flow (McCauley 1994; Powell et al.
© 1999 NRC Canada
18
1996b; Petit et al. 1997), introgressive hybridization events
(as exemplified by N. glutinosa and N. tabacum in the pres-
ent study), the origin of polyploids (Provan et al. 1997), and
last, but not least, maternal vs. paternal chloroplast inheri-
tance in disputed cases (Cato and Richardson 1996). RFLP
studies have indicated a strictly paternal mode of cpDNA in-
heritance 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 (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 study
comply with the current laws of New Zealand.
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