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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