J Appl Genet 46(1), 2005, pp. 41-43 Short communication Reciprocal controlled crosses between Pinus sylvestris and P. mugo verified by a species-specific cpDNA marker Witold Wachowiak 1, Andrzej Lewandowski 1, Wies³aw Prus-G³owacki 2 1 2 Polish Academy of Science, Institute of Dendrology, Kórnik, Poland Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz University, Poznañ, Poland Abstract. A species-specific marker of cpDNA (paternally inherited in pines) was used to verify the hybrid origin of seedlings from controlled reciprocal crosses between Pinus sylvestris and P. mugo. A very low degree of compatibility between those two species has been revealed. In the three consecutive years of experiments, no filled seeds were obtained in the combination with P. mugo as the seed parent. From P. sylvestris as the seed parent and P. mugo as the pollen donor, we succeeded to obtain four filled seeds (about 1 %), but only in one year. The seedling obtained from the seeds had cpDNA haplotypes specific to P. mugo, which proves their hybrid origin. This method enables verification of the result of controlled crosses. The importance of the results has been discussed in the aspect of postulated natural hybridisation in sympatric populations of the two species. Key words: controlled crosses, cpDNA marker, hybridisation, Pinus mugo, Pinus sylvestris. The Scots pine (Pinus sylvestris) is a widespread tree species and the main forest-forming component in Europe and Asia. P. sylvestris is closely related to the dwarf mountain pine (P. mugo), which is endemic to European mountainous regions and also occurs on some peatbogs from the postglacial period (Critchfield and Little 1966). Individuals with intermediate or mixed phenotypic characters with regard to P. sylvestris and P. mugo were found in sympatric populations of the species (Prus-G³owacki and Szweykowski 1980; Yurukov and Tashew 1992). Studies on their hybridisation were conducted through artificial crossing. These experiments were also carried out to determine phylogenetic relationships among pine species and for breeding purposes (Kormutak and Lanakova 1988; Prus-G³owacki and Stephan 1998). The production of filled seeds has mostly been used as a criterion of successful crosses. However, due to the lack of species-specific markers the hybrid origin of the seeds from these experiments and the individuals from sympatric populations has not been proved so far. Nowadays, the application of DNA markers of the plastid genome, which is paternally inherited in pines (Wagner 1992), enables verification of the results of controlled crosses. The comparative analyses of species-specific cpDNA haplotypes of paternal tree and F1 progeny make it possible to confirm hybridization. In the presented study a species-specific cpDNA marker was applied to verify the hybrid origin of the progeny from controlled crosses between P. sylvestris and P. mugo. The study aimed to check the crossability between the species in the aspect of their postulated natural hybridisation. Two individuals of P. sylvestris Received: November 26, 2004. Accepted: December 16, 2004. Correspondence: W. Wachowiak, Polish Academy of Science, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland; e-mail: witold.wachowiak@oulu.fi 42 W. Wachowiak et al. originating from the seed orchard in Zwierzyniec near Kórnik in Poland and two of P. mugo from the Arboretum of the Institute of Dendrology, Polish Academy of Science, were used in the reciprocal controlled crosses. One individual from each species was used as the pollen donor. Controlled crosses were carried out according to the procedure by Moulalis et al. (1976) in three consecutive years, from 1998 to 2000. In May, two weeks before the expected flowering, about 50 female strobili of each species were isolated in polyethylene bags when flower buds were large enough and the peduncle was visible. Two days before the expected pollination, male strobili were collected. Pollination was carried out during the fifth stage of the female strobili development, namely when cone scales were open and stood almost at a right angle to the axes of the strobili. The pollination was repeated 2–3 days later, depending on the prevailing weather conditions. The pollen was applied into the bags by a medicine dropper, with a rubber blower equipped with a valve. As a result, in the 3-year experiment on P. mugo as maternal individuals and P. sylvestris as the pollen donor, only 3 mature cones were obtained, all with undeveloped seeds. Conelet abortion took place mainly the following May, after pollination. In the crosses with P. sylvestris as maternal individuals, about 70% of conelets developed into mature cones but most of them contained empty or undeveloped seeds. Only in 2001, we succeeded to obtain 4 filled seeds. Two of them originated from one P. sylvestris tree, along with 254 empty seeds, whereas the other two, with 182 empty seeds, originated from the other individual. The method developed by Dumolin et al. (1995) was used to extract DNA from fresh needle material (50–100 mg) of the parental trees and from whole 3-week-old seedlings grown from the seeds obtained in the controlled crosses. We applied the previously described species-specific DNA marker of the trnL-trnF region (Wachowiak et al. 2000). This PCR-RFLP marker represents a DraI restriction site polymorphism in the above region, which leads to one band of PCR products for P. sylvestris and two bands for P. mugo. The digested PCR products are separated in a 2% (w/v) agarose gel and visualized under UV after staining with ethidium bromide. Figure 1 present the cpDNA haplotypes of the parental trees and their progeny. The four individuals obtained from the crosses with P. sylvestris as the seed tree and P. mugo as Figure 1. PCR products of trnL-trnF cpDNA region digestion with DraI enzyme in P. mugo (lanes 1–2); P. sylvestris (lanes 3–4); and P. sylvestris (&) × P. mugo (%) (lanes 5–8). M = 100-bp marker (Ladder Plus, Fermentas) the pollen donor displayed a cpDNA marker specific to P. mugo, which unambiguously indicates that they are hybrids. This result also proves the usefulness of cpDNA markers for identification of hybrids. The very low degree of crossability between these two species (about 1%) observed in this study is consistent with previous reports. Moulalis et al. (1976) obtained 31 full and germinating seeds by pollination of 309 female strobili from 7 P. sylvestris trees with P. mugo pollen. The results presented above, together with the results of other experiments (Kormutak 1990), prove that crosses between P. sylvestris as the seed parent and P. mugo as the pollen donor are possible in spite of the small efficiency of that process. Controlled crosses should be repeated for several years in order to formulate general conclusions concerning crossability of the studied species and to exclude the influence of the used provenances, biotic factors, and environmental conditions during and after pollination (Moulalis et al. 1976). Therefore, the absence of filled seeds in the reciprocal crosses conducted in the presented study does not exclude the possibility of hybridisation with P. mugo as the seed parent. Successful crosses in this combination were obtained in previous studies (Kormutak and Lanakova 1988). However, due to the lack of diagnostic markers, it was not possible then to confirm species identity of parental trees as well as the hybrid origin of seeds and to exclude uncontrolled pollination in the course of the experiments. P. sylvestris and P. mugo crossability The absence of diagnostic characters enabling identification of the hybrids has not allowed formulating so far any coherent concept of the postulated processes of P. sylvestris and P. mugo natural hybridisation. Therefore, the estimated frequency of hybridisation based on biometric and biochemical analyses ranges from rare formation of hybrids (Christensen and Dar 1997; Odrzykoski 2002) to formation of hybrid swarms (Staszkiewicz 1993). The presented results suggest a limited gene flow and existence of hybridisation barriers between these species. 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