2004, vol. 51, 67–72
Witold Wachowiak, Alina Bączkiewicz,
Konrad Celiński, Wiesław Prus-Głowacki
Species-specific chloroplast DNA
polymorphism in the trnV-rbcL region
in Pinus sylvestris and P. mugo
Received: 20 February 2004, Accepted: 26 March 2004
Abstract: Four cpDNA regions were analyzed with the use of PCR-RFLP technique and nucleotide sequences
of two mtDNA regions were characterized in order to find P. sylvestris and P. mugo species specific markers useful for studies of the species hybridization. The difference in the restriction fragment patterns of trnV-rbcL region after digestion with MvaI endonuclease was detected. The analyses of the species representatives from
various geographic regions revealed that the observed polymorphism is species specific. No differences have
been disclosed in the analyzed trnS-trnT, trnK1-trnK2, trnC-trnD cpDNA regions. The P. sylvestris and P.mugo
mtDNA sequences of orf25 and coxI regions proved to be identical.
Additional key words: Scots pine, dwarf pine, hybridisation, DNA markers, mtDNA,
Addresses: W. Wachowiak, Polish Academy of Science, Institute of Dendrology, ul. Parkowa 5, 62-035
Kórnik, Poland wachwit@amu.edu.pl
A.Bączkiewicz, Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz University,
Międzychodzka 5, 60-371 Poznań, Poland
K.Celiński, Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz University,
Międzychodzka 5, 60-371 Poznań, Poland
W. Prus-Głowacki, Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz University,
Międzychodzka 5, 60-371 Poznań, Poland
Introduction
The mechanism of uniparental inheritance of organelle genomes may be applied in the studies on hybridisation and introgression between closely related
forest tree species. In conifers, the plastid genome is
transmitted paternally whereas the mitochondrial genome in the maternal line (Neale et al. 1986; Neale
and Sederoff 1988). The use of species specific
chloroplast (cpDNA) and mitochondrial (mtDNA)
markers allows for identification of hybrids displaying the genomes from different species. These studies
also enable to define the direction and intensity of hybridisation and its influence on the genetic structure
of the species sympatric populations. The markers of
organelle genomes have been applied in the analyses
of hybridisation processes among the species of Abies
(Isoda et. al. 2000), Picea (Bobola et. al.1996; Perron
and Bousquet 1997; Germano and Klein 1999) or Pinus (Wagner et. al. 1987; Wang and Szmidt 1990).
Biometric and biochemical (isozymes) analyses of
the individuals from Scots pine (Pinus sylvestris) and
dwarf pine (P. mugo) sympatric stands have not resulted in formulating one coherent hypothesis about
68
Witold Wachowiak, Alina Bączkiewicz, Konrad Celiński, Wiesław Prus-Głowacki
the postulated processes of the species hybridisation
(Prus-Głowacki and Szweykowski 1983; Staszkiewicz
1993; Siedlewska and Prus-Głowacki 1994). The estimation of the hybridisation intensity vary from rare
formation of hybrids (Neet-Sarqueda et al. 1988;
Christensen and Dar 1997) to the formation of hybrid
swarms, consisting of F1 hybrids, hybrids of subsequent generations and introgressants (Staszkiewicz
and Tyszkiewicz 1969; Bobowicz 1990; Vievegh and
Cambalowa 1993). These different opinions result
from the absence of diagnostic features for particular
species, which would allow for certain hybrids identification. Considering the absence of biometrical and
biochemical diagnostic traits the application of
cpDNA and mtDNA markers specific to the analyzed
species seems to be a good approach aimed at verifying the hypotheses about the frequency and the direction of the species hybridization.
So far there have been described species specific for
P. sylvestris and P. mugo cpDNA markers in the trnF-trnL
region (Wachowiak et al. 2000). The present study is
the continuation of the analyses aiming at mtDNA
markers development and detecting additional differences in the cpDNA regions which would be useful to
prove the gene exchange among the species.
Methods
The P. sylvestris and P. mugo representatives used in
this study have been derived from different geographical regions (Table 1). P. sylvestris S1-S20 individuals
(apart from S9) come from the Scots pine 1982 provenience trail in the Institute of Dendrology, Polish
Academy of Science in Zwierzyniec near Kórnik, coordinated by IUFRO (International Union of Forest Research Organization). The trial consists of 20 populations representing the European range of P. sylvestris
(Boratyński 1991). The S9 P. sylvestris individual was
collected in Uścikuwiec. The M1-M14 P. mugo was derived from the populations in the Botanical Garden of
the Polish Academy of Science, in Warsaw – Powsin
and they originate from the natural stands in the
Tatra Mts. (Marczewski 1993). The plant material
from the remaining P. mugo individuals were collected
in their natural populations (Table 1).
The needles from trees (ca. 100 mg of fresh material) undergone DNA extraction using the method described by Dumolin et al. (1995). Four cpDNA and
two mtDNA regions were amplified with the use of
the primers described in Table 2. PCR was run in a
Personal Cycler (PTC-200, MJ Research, USA).
PCR-amplification was carried out in a total volume
of 25 µl containing about 20 ng of template DNA, 2.5
mM MgCl2, 100 µM of each dNTP, 0.2µM of each
primer and 0.25 U of Taq polymerase with the respective 1× PCR buffer (Taq polymerase and 10× PCR
buffer were purchased from Fermentas, Lithuania).
Preliminary denaturation of DNA for 5 min at 95°C
was followed by 30 cycles of denaturation at 93°C for
30 sec, primer annealing for 45 sec at 57.5° (for
trnV-rbcL, trnC-trnD regions) or 53°C (for trnS-trnT,
trnK1-trnK2, orf25 and coxI regions), DNA synthesis
at 72°C for 2 min for cpDNA and 1 min for mtDNA
primers. The reaction was terminated with 10 min incubation at 72°C.
PCR-RFLP analyses of the amplified cpDNA regions were performed with the use of restriction enzymes described in Table 3. The enzymes applied for
digestion of each region were derived in a random selection. Two P. sylvestris and two P. mugo individuals
were chosen for the restriction analyses. In case of detecting the interspecies specific differences of the investigated regions the remaining individuals used in
the study were also analysed. 10 µl of PCR product
underwent digestion, following the reaction conditions recommended by the enzyme provider
(Fermentas, Lithuania). After digestion, the samples
were separated in 8 % polyacrylamide gel (Sambrook
et al. 1989), stained with ethidium bromide and analysed by UV light.
The PCR product of orf25 and cox1 mtDNA were
purified with the use of QIAquickTMPCR Purification
Kit (Qiagen) and sequence characterized in one P.
sylvestris (S12) and one P. mugo (M8) individuals. Sequencing was performed through automatic sequencer (PerkinElmer) with the use of Big Dye Terminator DNA Sequencing Kit (Applied Biosystems)
according to a protocol by Liepelt et al. (2001).
Results
The PCR products of orf25 and cox1 mtDNA regions have a length of about 500 bp and 700 bp, respectively. The sequences of orf25 mtDNA region were
submitted
to
Gene
Bank
database
(http://www.ncbi.nlm.nih.gov) and they have the
following accession numbers: AF361058 for P. mugo
and AF361059 for P. sylvestris. The P. sylvestris sequences of coxI and orf25 regions have proved no difference comparing to the corresponding sequence of
P. mugo. The coxI sequences of both species were identical with the coxI sequence of P. sylvestris from Gene
Bank database (AJ000354).
The amplification products of trnV-rbcL, trnS-trnT,
trnK1-trnK2, trnC-trnD cpDNA regions have the
length of about 4000 bp, 1700 bp, 2200 bp and 2000
bp, respectively. The PCR-RFLP analyses of trnV-rbcL
cpDNA region digested with the use MvaI
endonuclease indicated the length differences of the
obtained DNA fragments between P. sylvestris and P.
mugo. The length of fragments differentiating both
species is about 400bp and 500bp for P. sylvestris, and
440bp and 460bp for P. mugo. All the analysed P.
sylvestris individuals (Table 1) have displayed type S
Species-specific chloroplast DNA polymorphism in the trnV-rbcL region ...
69
Table 1. The origin and number of the analysed P. sylvestris and P. mugo individuals
No.
The origin
Country
Latitude
Longitude
Meters above sea
level
Number of analysed trees
Pinus sylvestris
S1
Roshchinskaya Dacha Russia
60°15’
29°54’
80
1
S2
Kondezhskoe
Russia
59°58’
33°30’
70
1
S3
Serebryanskoe
Russia
58°50’
29°07’
80
1
S4
Silene
Latvia
55°45’
26°40’
165
1
S5
Miłomłyn
Poland
53°34’
20°00’
110
1
S6
Supraśl
Poland
53°12’
23°22’
160
1
S7
Spała
Poland
51°37’
20°12’
160
1
S8
Rychtal
Poland
51°08’
17°55’
190
1
S9
Uścikuwiec
Poland
52°40’
16°50’
90
1
S 10
Neuhaus
Germany
52°24’
13°54’
40
1
S 11
Betzhorn
Germany
53°02’
10°30’
650
1
S 12
Lampertheim
Germany
52°30’
10°00’
95–100
1
S 13
Ardennes
Belgium
50°46’
4°26’
110
1
S 14
Haguenau
France
48°49’
7°47’
130–180
1
S 15
Sumpberget
Sweden
60°11’
15°52’
185
1
S 16
Zahorie
Slovenia
48°46’
17°03’
160
1
S 17
Pornoapati
Hungary
47°20’
16°28’
400
1
S 18
Maocnica
Serbia
43°10’
19°30’
1200
1
S 19
Prusacka Rijeka
Bosnia
44°05’
17°21’
800–970
1
S 20
Catacik
Turkey
40°00’
31°10’
1380–1420
1
Pinus mugo
M1-M14 Tatra Mts.
Poland
58°35’
14°45’
1700
14
Śn 1–3
Sudet Mts.
Poland
50°49’
15°46’
1500
3
U 1–21
Urlea
Romania
24°80’
45°70’
2470
2
Table 2. The PCR primer sequences of the analyzed cpDNA and mtDNA regions
cpDNA region
5’–3’ sequence of primer 1
5’–3’ sequence of primer 2
References
trnV-rbcL
cga acc gta gac ctt ctc gg
gct tta gtc tct gtt tgt gg
Dumolin et al. 1997
trnS-trnT
cga ggg ttc gaa tcc ctc tc
aga gca tcg cat ttg taa tg
Demesure et al. 1995
trnK1-trnK2
ggg ttg ccc ggg act cga ac
caa cgg tag agt act cgg ctt tta
Demesure et al. 1995
trnC-trnD
cca gtt caa atc tgg gtg tc
ggg att gta gtt caa ttg gt
Demesure et al. 1995
mtDNA region
5’–3’ sequence of primer 1
5’–3’ sequence of primer 2
References
orf25
atg cta ttt gct gct att cc
agg act atc aag cct tct cg
Wang et al. 1996
coxI
tta tta tca ctt ccg gta ct
agc atc tgg ata atc tgg
Glaubitz and Carlson 1992
haplotype, whereas all the P. mugo individuals have
possessed type M haplotype (Fig. 1). PCR-RFLP analysis of trnV-rbcL region, which applied the other restriction enzymes, as well as the analysis of the
trnS-trnT, trnK1-trnK2, trnC-trnD cpDNA regions presented in Table 3, have not shown any differences of
the restriction patterns between P. sylvestris and P.
mugo.
Discussion
The high homology of the plastid genome of higher
plants, which has been supported by the comparative
analyses of the complete cpDNA sequences of several
species, has enabled to describe some universal PCR
primers allowing the amplification of the corresponding DNA regions even in the group of phylogenetically
distinct taxa (Taberlet et al. 1991; Demesure et al.
1995). The four previously described pairs of PCR
primers designed for the amplification of non-coding
regions between tRNA genes have been applied in the
presented study. Since the level of mutation observable in these regions is significantly higher than in conservative coding sequences, these regions have been
regarded as particularly useful ones in the analyses of
species variability (Dumolin-Lapegue et al. 1997).
70
Witold Wachowiak, Alina Bączkiewicz, Konrad Celiński, Wiesław Prus-Głowacki
Fig. 1. 8.0 % polyacrylamide gel of PCR products as amplified from the chloroplast DNA region trnV-rbcL and digested with the restriction enzyme MvaI. P. sylvestris
individuals (S3, S18) exhibit a distinct different banding
pattern (haplotype S) as compared with the P. mugo individuals (M3, M14) (haplotype M). Right line of the gel –
molecular size standard (Smart-Ladder, Eurogentec,
Germany)
The PCR-RFLP analyses conducted with the application of different restriction enzymes have resulted
in detecting the polymorphism of trnV-rbcL region
differentiating P. sylvestris and P. mugo. The observable
differences in the electrophoresis migration rate of
the obtained DNA fragments may be correlated with
the presence of point mutations in two sites of the
species sequences which cause the loss/gain of the restriction site for MvaI enzyme. The existence of the
restriction sites in different parts of the amplified regions of both species leads to DNA fragment length
polymorphism and does not influence the number of
obtained fragments.
The identical restriction patterns for the other analysed cpDNA regions indicate the absence of deletion/insertion differences which could be detected by
the applied DNA electrophoresis in polyacrylamide
gel. In these regions, however, there can still occur
Table 3. cpDNA regions conducted to the PCR-RFLP analyses
The amplified
cpDNA regions
The applied restriction enzymes
trnV-rbcL
MboI, MspI, MvaI, PstI, SmaI
trnS-trnT
AluI, Bsh1236I, BsuRI, DraI, HinfI, Hin6I, RsaI
Sau3A
trnK1-trnK2
Bsp120I, Eco32I, HindIII, MboI, MspI, MvaI, PstI
trnC-trnD
AluI, ApaI, Bsp120I, BsuRI, DraI, EcoRI, Eco32I,
Eco471, HaeIII, Hin6I, HindIII, HinfI, KpuI, MboI,
MspI, PstI, RsaI, SacI, SmaI
nucleotide substitution that have not been discovered. The comparative analyses of cpDNA sequences
of numerous Pinaceae representatives indicate the relatively high rate of nucleotide substitution in the
inter genic regions in relation to the parts of coding
regions subjected to stronger selective effects
(Ziegenhagen and Fladung 1997; Wang et al. 1999).
In spite of the conservative character of genes and a
relatively low level of the sequence polymorphism in
the group of even phylogenetically distinct taxa, the
cases of intra-species variability of cpDNA regions
have been detected, among others, for Abies alba
(Ziegenhagen et al. 1995), Quercus robur (Dumolin et
al. 1995), Fagus sylvatica (Demesure et al. 1996) or
Nothofagus nervosa (Marchelli et al. 1998). In the present study, the restriction analysis of the trnV-rbcL region carried out with the use of MvaI enzyme for individuals from different geographical regions has
proved the presence of S haplotype in all P. sylvestris
individuals and M haplotype in all P. mugo individuals.
This result indicates that the observed differences are
species-specific and allow for distinguishing P.
sylvestris from P. mugo based on plastid DNA.
In spite of the highly conserved character of the
mtDNA sequences in higher plant, the examples of
nucleotide substitutions in the coding regions were
indicated. Due to the observable mechanism of RNA
editing these differences do not effect the changes of
the peptide amino acids component and do not undergo the high selection influences (Glaubitz and
Carlson 1992; Wang and Szmidt 2001). Therefore,
the PCR-RFLP analyses of the orf25 and coxI mtDNA
regions conducted before, which have not indicated
the sequence differences between P. sylvestris and P.
mugo, have not excluded the existence of undetected
point mutations (Wachowiak et al. 2000). Nevertheless, the presented results indicate that the P. sylvestris
sequences of these regions are identical with the corresponding sequences of P. mugo. Thus, these regions
are not useful for developing the species specific
mtDNA markers.
The differences in the trnV-rbcL region detected in
this study together with previously described for P.
sylvestris and P. mugo cpDNA species-specific markers
proved to be useful in the hybridisation analyses of
both species. Paternally transmitted cpDNA markers
particularly allow indicating the present episodes of
hybridisations in case of the detected inconsistency of
the species-specific cpDNA haplotypes of the parental
tree and its F1 progeny. Nevertheless, it is still necessary to develop species specific mtDNA markers,
which would allow for more precise analyses of the
species hybridisation and identification of hybrid
trees.
Species-specific chloroplast DNA polymorphism in the trnV-rbcL region ...
Conclusions
1) The PCR-RFLP analyses allow indicating the differences between P. sylvestris and P. mugo in the
trnV-rbcL cpDNA region digested with the use of
MvaI restriction enzyme
2) The analyses of the species representatives from
different geographical regions have proved that the
observed differences in the trnV-rbcL region are
species-specific and useful in the studies of hybridisation between P. sylvestris and P. mugo
3) No deletion/insertion or nucleotide substitutions
were detected in the trnS-trnT, trnK1-trnK2 and
trnC-trnD cpDNA regions of P. sylvestris and P. mugo
4) No differences were detected in the corresponding
sequence of orf25 and coxI mtDNA of P. sylvestris
and P. mugo
Acknowledgements
Thanks are given to Sascha Liepelt from Nature
Conservation Division, Faculty of Biology,
Philipps-University Marburg, Germany for his help in
DNA sequencing. The research was supported by the
State Committee for Scientific Research, Poland
(KBN Grant no. 0306/P04/2001/21).
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