Art11 Journal of Applied Botany and Food Quality 81, 165 - 171 (2007) 1Institute of Forest Genetics and Forest Tree Breeding, Georg-August University Göttingen, Germany 2Landesbetrieb Wald und Holz Nordrhein-Westfalen, Außenstelle Arnsberg Forstgenbank NRW Chloroplast DNA analysis in oak stands (Quercus robur L.) in North Rhine-Westphalia with presumably Slavonian origin: Is there an association between geographic origin and bud phenology? O. Gailing1, H. Wachter2, J. Heyder2, H.-P. Schmitt2, R. Finkeldey1 (Received August 6, 2007) Summary Slavonian oaks (Quercus robur subsp. slavonica) have been intro- duced into Germany in the second half of the 19th century from the lowlands of the rivers Save and Drava in today’s Croatia. If compared to indigenous oak stands, they are characterized by good growth, comparatively low seed production and a late bud burst. Based on the information of European-wide variation patterns at chloroplast DNA markers in oaks we adapted chloroplast microsatellites for the analysis of all oak stands of presumably Slavonian origin in the Münsterland and lower Rhine regions. We were able to distinguish between Slavonian haplotypes with no natural occurrence in the study area and indigenous types that do not occur in the Balkan region. A generally high differentiation among stands was observed at chloro- plast markers (GST = 0.674). Based on the haplotype information and historic records we found that stands with Slavonian material have been established between the years 1878 and 1903. In a total of 910 analysed trees the Slavonian haplotypes 5, 2 or 17 were the most frequent ones but a considerable amount of samples with indigenous haplotype 1 or haplotype10 with presumed origin in Southwestern Europe was also present. A clear association between haplotype 2 and late bud burst was detected in adult stands and in a field trial established with seeds from Slavonian and indigenous oak stands. The information about the haplotype composition in all Slavonian stands can be used as reference for the certification of reproductive material. The analysis of cpDNA haploytpes in old oak stands that had been established before the introduction of foreign seed material can give valuable information for the identification of indigenous oak stands. Introduction Slavonian pedunculate oaks had been introduced into Germany (Münsterland, North Rhine-Westphalia) in the second half of the 19th century when extensive seed trade by railway started. Historical documents and genetic marker analyses using chloroplast DNA indicated that Slavonian oaks originated from the lowlands of the rivers Save and Drava in today’s Croatia (GAILING et al., 2007; GEHLE, 1999; HESMER, 1955; WACHTER, 2001). Slavonian oaks in Germany are characterized by late bud burst, fast growth and long clear boles when compared to indigenous oaks that had been planted at the same time in the same stand (GAILING et al., 2003; WACHTER, 2001). Earlier studies indicated that stands established with Slavonian oaks flushed two or three weeks later than neighboring indigenous oak stands, possibly resulting in better resistance to late frosts and lower susceptibility to pests (e.g. feeding damage by the European oak leaf roller Tortrix viridana) (GAILING et al., 2003; WACHTER, 2001). However, the number of presumably Slavonian stands that have been characterized at chloroplast DNA markers and for bud phenology was rather limited. Chloroplast DNA (cpDNA) markers can give valuable information about the geographic origin of oaks and other angiosperms. The chloroplast genome is uniparentally inherited in maternal lineages; usually, recombination does not occur. Thus, patterns of seed dispersal can be recognized at cpDNA markers (DUMOLIN-LAPÈGUE et al., 1998). An European-wide inventory of more than 2600 populations of white oaks using PCR-RFLPs of four non-coding chloroplast regions (PETIT et al., 2002b) revealed a total of 45 haplotypes with a characteristic distribution in Europe (PETIT et al., 2002b). In a former study we adapted chloroplast microsatellites (DEGUIL- LOUX et al., 2003; WEISING and GARDNER, 1999) for the analysis of oak stands in the Münsterland region. By applying these markers we were able to distinguish most chloroplast haplotypes and found complete congruence with the PCR-RFLP procedure (GAILING et al., 2007). These and an additional new marker were used in the present study in order to determine the haplotype composition of all putative Slavonian oak stands in North Rhine-Westphalia (Germany). Three control stands were included in the analysis that have been identified to be indigenous according to their growth habit, bud phenology and /or due to the early stand establishment before the introduction of Slavonian oaks into Germany. Here, we present the results of the cpDNA analysis of 33 newly investigated stands. Haplo- type frequency and diversity within and among stands were analysed by including data from 17 formerly analysed stands (GAILING et al., 2003; GAILING et al., 2007). The aims of the present study are to give an overview of the haplotype composition of all pedunculate oak stands with presumably Slavonian origin in North Rhine-Westphalia and to determine the time frame in which stands with different origins (haplotypes) have been estab- lished. More specific aims are the development of reliable and easy- to-use genetic markers as a basis for the certification of reproductive material of Slavonian oaks in Germany and to lay a basis to distin- guish between indigenous and introduced plant material. Finally, we want to test for an association between geographic origin of oaks (identified by the chloroplast haplotype) and the putatively adaptive character bud burst. Materials and methods Plant material A total of 50 stands (mean of 18.2 samples per population) of Quercus robur in the Münsterland and from the Lower Rhine region have been studied including all putative stands of Slavonian oak (Quercus robur ssp. slavonica) in North Rhine-Westphalia (Tab. 4). Most of them have been characterized as Slavonian oaks according to their growth habit, bud phenology (late flushing) and according to his- torical documents. Samples from one stand (Croatia) were collected directly in Croatia (Vinkovsi). Three of the stands were characterized to be indigenous Q. robur according to growth habit and historic documents and were used as references. Seventeen of the stands were included in earlier studies (GAILING et al., 2003; GAILING et al., 2007). Additionally, four provenances of presumably Slavonian origin grown in a field trial in Dormagen (Abt. 33C/F, Frh. v. Nagel-Doornick; Abt. 35C1; Abt. 161 A1, Frh. v. Boeselager; Abt. 343a3, FA Peine) and a reference stand (Frh. v. Vittinghoff-Schell) were characterized at chloroplast DNA markers and for bud phenology (20 trees per 166 O. Gailing, H. Wachter, J. Heyder, H.-P. Schmitt, R. Finkeldey provenance). The field trial was established in 1993 with seedlings grown from seed material that was collected in stands in 1988. DNA isolation Total genomic DNA was extracted from fresh leaves (a small slice of about 1 cm2) or from buds with the DNeasy Plant Kit for 96 probes from Qiagen (Hilden, Germany). The amount of DNA was checked on 1% agarose gels after staining with ethidium bromide. PCR-RFLP technique The following non-coding regions of the chloroplast genome trnD- trnT with TaqI (DT), trnC-trnD with TaqI (CD), psa-trnS with Hinf I (AS) were studied with the PCR-RFLP technique (DEMESURE et al., 1995). The PCR profile was as described by DEMESURE et al. (1995). PCR amplification was performed in a 15 µl volume containing about 10 ng template DNA, 1x Qiagen PCR buffer; 1x Q-solution (Qiagen), 2 mM of MgCl2, 0.20 mM each dNTP (Fermentas); 0.5 µM of each primer and 1U Taq DNA polymerase (Qiagen). The restriction re- actions were performed by adding 5 µl of PCR product to a mix containing 3.5 µl H2O, 1.0 µl 10x enzyme buffer (Roche) and 5 units of the enzyme. The reactions were incubated for 5h at 37°C (HinfI) and at 65°C (TaqI). The restriction fragments were separated on a 8% polyacrylamid (PAA) gel, stained with SYBR Gold (Molecular probes) and visualized under UV light. The interpretation of the re- striction pattern followed PETIT et al. (2002b). Control probes were used to assign the resulting patterns to haplotypes described in the European-wide inventory of oak species at cpDNA markers. Chloroplast microsatellites Thirteen chloroplast microsatellites developed for dicotyledonous angiosperms (WEISING and GARDNER, 1999) or specifically for oak (DEGUILLOUX et al., 2003) were tested for polymorphisms (GAILING et al., 2007). Additionally one cpSSR marker (odt) has been developed from sequencing the trnD-trnT region of the chloroplast genome. The sequencing reactions were carried out with the Big Dye termi- nator v. 1.1 Cycle sequencing kit (Applied Biosystems). Primers were designed using the software primer 3 (ROZEN and SKALETSKY, 2000). In total, six cpSSRs were polymorphic in our sample (Tab. 1). Two of these markers (ucd4, udt4) were sufficient to distinguish between all main haplotypes detected by the PCR-RFLP method (GAILING et al., 2007). Only H5 and H7-26 could not be distinguished by cpSSRs. However, they were easily distinguishable by amplification of the trnD-trnT region and restriction with TaqI. Differences in the restriction pattern were clearly recognized on a 2% agarose gel. Data analysis As in most angiosperms the chloroplast genome in oaks is maternally inherited reflecting the dispersal by seeds (DUMOLIN-LAPÈGUE et al., 1998). Since it is equivalent to a single haploid locus the following analyses are based on the haplotype. Haplotypes were determined on the basis of the combination of different chloroplast microsatellite markers (see Tab. 2). Absolute haplotype frequencies were determined in each stand. Haplotype frequencies were used as input to calculate within-population diversity (HS) and total diversity of haplotypes (HT) in the program RAREFAC (PETIT et al., 1998). Likewise, allelic rich- ness as a measure adjusting for different sample sizes was calculated after rarefaction in the program RAREFAC (PETIT et al., 1998) with a rarefraction size equalling the smallest sample size. GST ((HT-HS)/ HT) was calculated for the partitioning of diversity among stands. RST was also calculated as a similar measure of population differen- tiation that is derived from estimates of allelic richness (PETIT et al., 1998). Assessment of bud burst Bud burst was scored on a scale ranging form 0 (buds closed) to 5 (leaves fully expanded). When samples were collected in the mid of May 2006, stands with extreme phenotypic values for bud burst were assessed (all trees of a given stand without leaves, or all leaves fully expanded). The experimental plot in Dormagen has been established with seed- lings from four different stands of presumably Slavonian origin and one reference stand (Vittinghoff-Schell) in year 1993. Pronounced differences in the dating of bud burst have been detected with late and early flushing trees growing side by side. Early and late flushing trees were randomly selected on April 30th in 2007. Bud phenology was assessed for individual trees using a scale from 0 (buds closed) to 5 (leaves fully expanded). The haplotype of each tree was deter- mined as described above. Results PCR RFLPs Combining the information from chloroplast regions CD, DT and AS a total of 7 haplotypes could be distinguished that corresponded Tab. 1: Characterization of chloroplast microsatellites locus name primer sequences (5’-3’) repeat size (bp) na Ta (°C) location author udt4 FAM-GATAATATAAAGAGTCAAAT (A)9 144-145 2 touchdown Intergenic trnE-trnT Deguilloux et al. 2003 CCGAAAGGTCCTATACCTCG ucd4 FAM-TTATTTGTTTTTGGTTTCACC (T)12 93-96 4 touchdown Intergenic ycf6-psbM Deguilloux et al. 2003 TTTCCCATAGAGAGTCTGTAT ukk4 HEX-TTGTTTACCTATAATTGGAGC (T)9 109-110 2 53 Intergenic matK-trnK Deguilloux et al. 2003 TAGCGGATCGGTTCAAAACTT ccmp2 FAM-GATCCCGGACGTAATCCTG (A)11 233-234 2 50 5‘to trnS Weising and Gardner ATCGTACCGAGGGGTTCGAAT 1999 ccmp10 HEX-TTTTTTTTTAGTGAACGTGTCA (T)14 111-112 2 52.5 Intergenic rp12-rps19 Weising and Gardner TTCGTCGDCGTAGTAAATAG 1999 odt FAM-GAGCGTCTCGCAAATTGTTA (A11) 228-229 2 51 Intergenic trnD-trnT this study TTACCGCTGTTCATTTGCTC Chloroplast DNA analysis of Slavonian oak stands 167 to haplotypes H1, H2, H4, H5, H7-26, H10* and H17 of the Euro- pean-wide inventory (PETIT et al., 2002b). Haplotypes 7 and 26 (H7- 26) are closely related most likely due to a post-colonisation mutation event (PETIT et al., 2002a) and and are summarized as H7-26 in the following. The comparison with the distribution of haplotypes in Europe showed that H1, H4 and H10* are described for the study area in Germany (Münsterland and Lower Rhine region) but are missing in the Balkan region (BORDÁCS et al., 2002; KÖNIG and STAUBER, 2004; KÖNIG et al., 2002; PETIT et al., 2002b). H1 is the most frequent type in the western part of Germany and dominant in central Europe. It had its glacial refugia most likely in southern Italy (PETIT et al., 2002a). H10* reveals a center of distribution in the southwest and west of Europe with presumed refugia on the Iberian Peninsula. H4 rarely occurs, mainly in Germany, Hungary, Poland and Romania (PETIT et al., 2002b). H2 and H17 are frequent in Croatia but do not occur naturally in Germany. H5 has a center of distribution in the Balkan region but is comparatively rare in Germany. In western Germany natural popu- lations with this haplotype are apparently missing (KÖNIG and STAUBER, 2004). H5 was also found in all samples of the reference stand from Croatia (Tab. 4) H2, 5, 7-26, 17 have a center of distribution in the Balkan region (BORDÁCS et al., 2002; PETIT et al., 2002b) but none of the haplotypes is restricted to this region. While haplotype H7-26 is mainly con- centrated in low mountain ranges in the western part of Croatia, haplotypes 2, 5, and 17 occur in the lowlands of the river Save between Zagreb and the Serbian border. H2 was also found in the Dinarides in Croatia. Only haplotype 7-26 occurs both in natural stands in the study area (KÖNIG and STAUBER, 2004), and in popula- tions from the Balkan region, but it is comparatively rare (n= 20) in the studied stands. Identification of haplotypes with cpSSRs All but haplotypes H5 and H7-26 could be distinguished with the combination of chloroplast microsatellites ucd4 and udt4 (Tab. 2). These two variants can be separated by PCR-RFLPs of trnD-trnT with TaqI. The results of the cpSSR method were fully congruent with the results obtained from the PCR-RFLP procedure (see also GAILING et al., 2007). Additionally, two variants of what was hitherto haplotype 5 were identified at chloroplast microsatellite odt (Tab. 2). Both types showed about the same frequency in the analysed stands. Most of the variation of those two types is distributed among populations (GST =0.72, calculated for populations where H5 is dominating). A higher fre- quency of one or the other type in stands with predominant haplo- type 5 is indicated by asterisks in Tab. 4. Variation within and among stands at cpDNA markers Complete haplotype information has been obtained for a total of 910 adults trees in 50 stands. The most frequent haplotype was H5 (n = 405) followed by H2 (n = 169), H1 (n = 154), H10* (n = 93), H17 (n = 54), H7-26 (n = 21) and H4 (n = 14). Most stands either showed only one haplotype (15 stands) or they are characterized by one predominant haplotype, a result that is reflected by high dif- ferentiation values among stands (GST = 0.674, RST = 0.734). The mean haplotype diversity and allelic richness within stands was 0.243 and 1.85, respectively. Thirteen stands had a haplotype diversity higher than 0.5 (probability to encounter two different haplotypes in a stand) due to a mixture of two or more (up to four) haplotypes per stand. Stands with predominant haplotypes 2 or 5 show the lowest haplotype diversity and allelic richness when compared to stands with other dominating haplotypes. Haplotypes 2 and 5 were the predominant types in eight and 20 stands, respectively. The indigenous haplotype 1 was the most frequent variant in six stands, and three additional stands (controls) were fixed on this type. This indigenous haplotype was detected in several stands in low frequency (Tab. 4). The oldest stand analysed established between 1826 and 1864 when seed transfer was less extensive revealed the indigenous haplotype 1. The Slavonian haplotypes 2 and 5 occurred in stands established between 1880 and 1895 as predominant types (Tab. 3). Another Slavonian type (H17) that is not indigenous to Germany occurred as the dominant type in six stands established between 1886 and 1912, the most recent one (Kottenforst 154B, 1912) was established from nursery-produced seedlings. A long period of time during which a stand had been established was not always associated with high haplotype diversity. For example, stand 76A (Cappenberg) was fixed on the indigenous type 1 and had been established between 1826 and 1864. However, while nine out of 20 stands established by direct sowing were fixed on only one haplotype, the five stands established from young nursery-produced seedlings showed a mixture of haplotypes reflected in higher haplotype diversity (HS = 0.468 (0.279-0.611)) as compared to stands established from seeds (HS = 0.232 (0 - 0.659)). Association of cpDNA haplotypes with bud burst Late bud burst was mainly observed in stands with predominant haplotype 2 (Tab. 4). In stand „Steprath“ trees with haplotypes 1, 10 Tab. 2: Characterization of chloroplast haplotypes with microsatellites. The size of the fragments is given in base pairs (bp) Haplotype ucd4 udt4 ukk4 odt ccmp2 ccmp10 1 95 145 110 228 233 112 2 93 145 110 228 233 111 4 95 144 109 229 233 112 5a 94 144 109 229 233 112 5b 94 144 109 228 233 112 7-26 94 144 109 229 233 112 10-11-12 95 143 109 228 234 111 17 96 145 109 228 234 111 ccmp2, ccmp10 (WEISING and GARDNER, 1999), ucd4, udt4, ukk4 (DEGUILLOUX et al., 2003), odt. Haplotype 5 und haplotype 7-26 can be distinguished by the primer-enzyme combination trnD-trnT TaqI. No distinction was made between southwest European types H10, H11 and H12 (designated as H10* in the text). Haplotypes 7 and 26 (H7-26) are closely related most likely due to a post-colonisation mutation event (PETIT et al., 2002a) and can not be distinguished with chloroplast microsatellites. 168 O. Gailing, H. Wachter, J. Heyder, H.-P. Schmitt, R. Finkeldey and 7-26 (n = 4) showed completely unfolded leaves in early May 2006, while buds of trees with haploytpe 2 were still mostly closed (n = 16). The same observation had been made in stands with mixed haplotype composition of individual trees in the Münsterland region (GAILING et al., 2003). Also in the experimental plot of Dormagen where Slavonian and indigenous oaks grow in the same environment, a strong association of Slavonian haplotype 2 with late bud burst was detected in early May 2007 (bud stage 3.56 ± 1.64, n = 32). Plants with haplotype 2 originating from stand 161A1 (Freiherr von Boeselager) revealed an even later bud burst (bud stage 2.43 ± 1.87, n = 12) than plants from stand 343a3 (Forstamt Peine, bud stage = 4.35 ± 0.81, n = 20). A few plants with Slavonian haplotype 5 show a slightly earlier bud stage (bud stage 4.70 ± 0.47, n = 27) than plants with indigenous haplotype 1 (n = 22) or southwest European haplo- type 10* (n = 9, all bud stage 5, leaves completely unfolded). Discussion Certification of reproductive material of Slavonian oaks Stands that had been established in the late 19th century with plant material from Croatia (Slavonian stands) show several favourable characteristics as fast growth, a long clear bole, and late bud burst that may result in a lower susceptibility to late frost and pest damage (WACHTER, 2001). On the other hand, seed set and natural regene- ration in Slavonian oaks in Germany is lower than in indigenous pedunculate oaks possibly due to the colder climate in the study area (lower annual mean temperatures) and/or restricted gene flow be- tween indigenous and Slavonian oaks. For example, it was shown that flushing dates of indigenous oaks and Slavonian oaks grown side by side are nearly non-overlapping (GAILING et al., 2003). How- ever, gene flow between Slavonian oaks and indigenous oaks and the effect on potentially adaptive characters as growth characteristics and seed production has not yet been studied. The data on the haplotype composition available for all oak stands of Slavonian origin lays the basis for the certification of reproductive material from these stands. Since cpDNA markers are maternally inherited in angiosperms with all seeds and natural regeneration showing the same chloroplast information as their mother tree, they show high uniformity within but a clear differentiation between stands. This is especially true for barochorous, wind-pollinated tree species such as oaks where seed dispersal is quite restricted if com- pared to dispersal by pollen. Thus, chloroplast markers are specifi- cally suited to test and falsify the origin of reproductive material of Slavonian and indigenous oaks. Since many stands of Slavonian oaks with a specific haplotype (H2) are uniform for favourable growth and stem characteristics and bud phenology (WACHTER, 2001), the analysis of seeds and young plants may allow to conclude on the phenotype of adult trees (see below experimental plot in Dormagen). However, gene flow by pollen from neighboring stands may affect potentially adaptive traits as bud burst (see below) or growth rate and other yield characteristics. The analysis of the highly informative cpSSRs markers allows the identification of the chloroplast haplotype even in older material or wood samples with highly degraded DNA (see below). An accurate distinction of small informative size differences can be performed in high-resolution capillary electrophoresis. By using internal standards an unambiguous distinction between chloroplast haplotypes is easily achievable (GAILING et al., 2007). Identification of Slavonian and indigenous oak stands Evidence for the establishment of stands with non-indigenous material is unambiguous for stands showing predominant haplo- types 2 or 17 that do not occur naturally in Germany. For stands showing predominant haplotypes with a wider distribution range in Europe a clear distinction between indigenous and introduced material based on cpDNA alone is not possible. Especially in Ger- many the diversity of different haplotypes is comparatively high due to a mixing of cpDNA lineages from different glacial refugia (PETIT et al., 2003). For example, H10* is most abundant in southwestern and western Europe but is a rarer occurrence in the study area. How- ever, for at least one stand with H10* the establishment with non- autochthonous material is well-documented (159A in Westtünnen). This stand is characterized by late bud burst and late leave fall, a combination of characters that is not found in indigenous and Slavonian stands in the same area and that may be interpreted as an adaptation to different climatic conditions in southwestern Europe (GAILING et al., 2003). Also H5 that was identified as the most common haplotype in the Slavonian stands of the study area does also naturally occur in Germany but according to historical documents and cpDNA analyses is most likely absent in natural stands of the study area (KÖNIG and STAUBER, 2004; KÖNIG et al., 2002). Since extensive seed transfer started with the expansion of railway con- nections in the second half of the 19th century, allochthonous oak populations are unlikely to be found among those established before 1860. The cpDNA analysis confirmed that seed material from Slavonian oaks was introduced only during the second half of the 19th century with the earliest occurrence of the Slavonian haplotypes H2 and H5 in 1880 and 1878, respectively. According to historical documents and cpDNA haplotype information Slavonian stands were established (from seeds) in Germany in the Münsterland and Lower Rhine region between 1878 and 1903. The further analysis of the haplotype composition of stands established before that time might help to identify cpDNA haplotypes that are characteristic for stands indigenous to the study area in the Münsterland and Lower Rhine regions. Since cpSSR primers amplify short informative regions of the Tab. 3: Estimate of haplotype diversity for stands with different dominating haplotypes Dominant year Year S N HS average HS range NA NArange haplotype dominant type H1 1826-1905 1826-1907 7 154 0.237 0 - 0.659 0.91 0 - 2.43 H2 1880-1894 1880-1894 8 166 0.175 0 - 0.363 0.78 0 - 1.76 H5 1880-1895 1878-1912 20 406 0.142 0 - 0.611 0.58 0 - 2.48 H7-26 1894 1881-1894 1 20 0.503 1.55 H10* 1886-1907 1880-1907 6 93 0.321 0 - 0.753 1.03 0 - 2.74 H17 1886-1912 1880-1912 6 54 0.541 0.485 - 0.602 1.42 1 - 1.90 HS: genetic diversity of haplotypes; NA: allelic richness, S: number of stands where the haplotype is dominating, N: total number of samples with the respective haplotype. H4 occurs in 14 samples and dominates in none of the stands. H4 occurred at low frequency in stands established between 1886 and 1912. Chloroplast DNA analysis of Slavonian oak stands 169 Tab. 4: Haplotype frequencies in Quercus robur stands Forest district stand year area planting/ collection chloroplast haplotypes (ha) sowing H1 H2 H4 H5 H7 H10* H17 FA Bergisch Gladbach, Königsforst 76B 1891 5.0 seeds buds closed May-06 0 19 0 0 0 0 0 H2 FA Bergisch Gladbach, Königsforst 127C 1887 3.4 seeds bud stage 5 May-06 0 0 0 20* 0 0 0 H5 FA Bonn, Kottenforst 10B2 1893 1.5 plants May-06 2 0 0 16 1 1 0 H5 FA Bonn, Kottenforst 37C 1907 2.2 seeds May-06 8 0 0 1 0 9 0 H10* FA Bonn, Kottenforst 40B 1907 2.2 seeds low growth May-06 0 0 0 0 0 19 0 H10* FA Bonn, Kottenforst 70D 1903 2.5 seeds May-06 0 0 0 9* 0 0 10 H17 FA Bonn, Kottenforst 85A 1912 3.7 plants bud stage 5 May-06 0 0 0 4 0 0 8 H17 FA Bonn, Kottenforst 85B 1905 4.1 seeds May-06 10 0 4 0 0 2 0 H1 FA Bonn, Kottenforst 85D 1904 8.6 seeds May-06 0 0 0 0 0 19 0 H10* FA Bonn, Kottenforst 89Aa 1904 8.4 seeds May-06 7 0 0 1 0 1 5 H1 FA Bonn, Kottenforst 95B 1903 2.8 seeds May-06 0 0 0 4 0 12 4 H10* FA Bonn, Kottenforst 134A/C 1902/1900 6.1 plants/seeds low growth May-06 5 0 6 2 0 7 0 H10* FA Bonn, Kottenforst 154B 1912 3.2 plants May-06 0 0 3 5 0 0 11 H17 Freiherr von der Leyen 17C 1885 7.1 ? bud stage 5 May-06 1 0 0 15* 0 3 0 H5 Fürst zu Salm-Salm, Rhede 105/4A1/A7 ? ? May-06 17 0 0 0 0 3 0 H1 Gut Ulenburg 4C ? 1.6 ? May-06 17 0 0 0 1 1 0 H1 Stadt Viersen 36B/38 1886 16.7 seeds May-06 0 0 1 18 0 0 0 H5 Steprath 3H 1881 0.5 ? buds May-06 1 16 0 0 1 2 0 H2 mostly closed Letmathe, H. Blix, Cappenberg Flur 2/155 1888 1.0 ? May-06 0 0 0 20** 0 0 0 H5 Letmathe, Estermann 116A1 1894 2.2 ? May-06 1 17 0 0 1 1 0 H2 Letmathe, Graf v. Kanitz 77C 1883 4.4 seeds May-06 2 0 0 18* 0 0 0 H5 Letmathe, Graf v. Kanitz 76A 1826-1864 9.5 ? May-06 20 0 0 0 0 0 0 H1 Letmathe, Graf v. Kanitz 19A 1883 1.6 seeds May-06 0 0 0 16* 0 0 0 H5 Letmathe, Graf v. Kanitz 32H/39A ? 2.0 ? May-06 0 0 0 20** 0 0 0 H5 Letmathe, Frhr. v. Boeselager 159A 1886 2.2 late bud burst, Apr-02 0 0 0 0 0 10 0 H10* late leave fall Letmathe, Frhr. v. Boeselager 159B 1887 3.3 early bud burst Apr-02 9 1 0 0 0 0 0 H1 Letmathe, Frhr. v. Boeselager 160B 1887 2.0 early bud burst Apr-02 10 0 0 0 0 0 0 H1 Letmathe, Frhr. v. Boeselager 161A1 1887 1.2 seeds late bud burst Apr-02 0 8 0 2 0 0 0 H2 Letmathe, Frhr. v. Boeselager 161A2 1879 1.0 seeds intermediate Apr-02 0 4 0 4 0 2 0 H2/H5 bud burst Letmathe, Schulze-Becking 54B1/B2 1880 1.5 May-06 0 6 0 13** 0 0 1 H5 Münsterland, BR Deutschland 1B1a ~1894 0.4 plants late bud burst Jul-05 0 17 0 1 2 0 0 H2 Münsterland, BR Deutschland 1B1b ~1890 0.5 plants intermediate Jul-05 2 4 0 12 0 0 2 H5 bud burst Münster, Ziebell, Lüdinghausen Flur 1/299 1880 4.0 ? May-07 0 19 0 0 0 1 0 H2 Obereimer, Gr. von Plettenberg 14D1 1890? 1.0 ? May-07 10 0 0 9 1 0 0 H1 Obereimer, Gr. von Plettenberg 104A 1895 1.8 ? May-06 1 0 0 19** 0 0 0 H5 Obereimer, Gr. von Plettenberg 106G 1888 2.9 ? May-06 0 0 0 19** 0 0 0 H5 Obereimer, VEBA 43 ~1894 0.7 ? Jul-05 0 18 0 1 1 0 0 H2 Warendorf, Frh. v. Nagel-Doornick 13A 1886 1.5 ? Jul-05 2 0 0 5 0 0 12 H17 Warendorf, Frh. v. Nagel-Doornick 24B ~1887 1.8 seeds Jul-05 0 0 0 17 1 0 0 H5 Warendorf, Frh. v. Nagel-Doornick 33C 1878 1.3 seeds Jul-05 0 0 0 20 0 0 0 H5 Warendorf, Frh. v. Nagel-Doornick 33D 1879 1.9 seeds Jul-05 20 0 0 0 0 0 0 H1 Warendorf, Frh. v. Nagel-Doornick 33F 1880 0.8 seeds Jul-05 0 0 0 20 0 0 0 H5 Warendorf, Frh. v. Nagel-Doornick 36E ~1882 1.5 seeds Jul-05 0 20 0 0 0 0 0 H2 Warendorf, Frh. v. Nagel-Doornick 50B ~1883 1.6 seeds Jul-05 2 0 0 17 0 0 0 H5 Warendorf, Schulze-Pellengahr 4H 1893 1.2 ? May-06 0 0 0 20** 0 0 0 H5 Warendorf, Schulze-Sutthoff Flur 2/77 ~1890 0.6 ? Jul-05 0 0 0 19 0 0 0 H5 Warendorf, Suttorp, Everswinkel Flur 11/129 ? 2.0 ? May-07 0 20 0 0 0 0 0 H2 Warstein-Rüthen, Kirche Anröchte 32C 1894 4.2 ? May-06 5 0 0 1 12 0 0 H7 Westkirchen, Quante Flur 2/90-92 ~1889-1890 2.2 seeds Jul-05 2 0 0 17 0 0 1 H5 Croatia, Vinkovsi 2007 May-06 0 0 0 20** 0 0 0 H5 sum haplotypes 154 169 14 405 21 93 54 a: samples were collected in the southwestern part of the stand. H7 is a shortcut for H7-26. Stand were either established by direct sowing (seeds) or from nursery-produced seedlings (plants). *: higher frequency of H5a **: higher frequency of H5b 170 O. Gailing, H. Wachter, J. Heyder, H.-P. Schmitt, R. Finkeldey chloroplast genome that are present in high copy numbers per cell in comparison with nuclear DNA markers, those markers can also be analysed in highly degraded samples (for example old wood samples) (DEGUILLOUX et al., 2004; RACHMAYANTI et al., 2006). The analysis of old and / or prehistorical wood samples at these markers might give additional information on the haplotype composition in oak stands before extensive long-distance seed transfer started. More recent studies indicate that authentic DNA can be retrieved from wood samples up to 1,000 years of age (LIEPELT et al., 2006). Association between haplotype and phenotype Slavonian provenances with haplotypes 2 and 5 of pedunculate oak showed an up to three week later bud burst than neighboring indi- genous oak stands in the Münsterland region (GAILING et al., 2003). These differences were found in four consecutive years where trees with H2 showed an even later bud burst than those with H5 (GAILING et al., 2003). In the present study bud burst has been assessed on a fixed date (April 30th) in a field trial in Dormagen containing plants with Slavonian, indigenous and southwest European haplotypes. While a strong association of haplotype 2 with later bud burst was detected, only minor differences in flushing date were recorded between individuals with Slavonian haplotype 5 and non-Slavonian haplotypes, possibly due to the assessment of the trait relatively late in spring. Progenies from stand 161A1 (Freiherr von Boeselager) with uniform late bud burst (GAILING et al., 2003) also showed the latest bud burst in the field trial reflecting a putatively strong genetic component (heritability) of the trait. Since also other stands with Slavonian haplotypes show a uniformly later bud burst than neigh- boring indigenous oak stands (GAILING et al., 2003; GAILING et al., 2007; WACHTER, 2001), underlying genetic differences have possibly evolved in response to different environmental (climatic) conditions in the regions of origin. While chloroplast markers can indicate geo- graphic origin, they are obviously not directly involved in the control of bud burst. Thus, due to the wide geographic distribution of most haplotypes, a prediction of the phenotype only from the haplotype information is in most cases not possible. Accordingly, KREMER et al. (2002) found in general no significant impact of chloroplast haplotype (maternal origin) on phenotypic traits such as bud burst in a test with 16 provenances of Q. petraea. The association between haplotype and late bud burst found for Slavonian stands in this study is most likely due to the fact that Slavonian oaks had been introduced into Germany from a restricted area in the lowlands of the rivers Save and Drava with similar environmental conditions. In order to resolve the genetic basis of adaptive differences in bud burst, an intra- specific crosses between adults trees of Slavonian and German origin with pronounced differences in flushing date have been produced. QTLs (Quantitative Trait Loci) will be located on genetic linkage maps calculated in a progeny of 192 full-sibs (GAILING, submitted). Acknowledgements We wish to thank Mrs. Olga Artes and Mrs. O. Dolynska for the technical help in the laboratory. For the help in collecting the plant material we thank Mrs. L. Schulze, Mr. Rogina. References BORDÁCS, S., POPESCU, F., SLADE, D., CSAIKL, U., LESUR, I., BOROVICS, A., KÉZDY, P. et al., 2002: Chloroplast DNA variation of white oaks in the northern Balkans and in the Carpathian Basin. Forest Ecol. Manage. 156, 197-209. DEGUILLOUX, M.-F., DUMOLIN-LAPEGUE, S., GIELLY, L., GRIVET, D., PETIT, R.J., 2003: A set of primers for the amplification of chloroplast micro- satellites in Quercus. Mol. Ecol. Notes 3, 24-27. DEGUILLOUX, M.-F., PEMONGE, M.-H., PETIT, R.J., 2004: DNA-based control of oak wood geographic origin in the context of the cooperage industry. Ann. Forest Sci. 61, 97-104. DEMESURE, B., SODZI, N., PETIT, R., 1995: A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 4, 129-131. DUMOLIN-LAPÈGUE, S., PEMONGE, M.-H., PETIT, R., 1998: Association between chloroplast and mitochondrial lineages in oaks. Mol. Biol. Evol. 15, 1321- 1331. GAILING, O., 2007: QTL analysis of leaf morphological characters in a Quercus robur full-sib family (Q. robur x Q. robur subsp. slavonica). Plant Biology (submitted). GAILING, O., WACHTER, H., LEINEMANN, L., HOSIUS, B., FINKELDEY, R., SCHMITT, H.-P., HEYDER, J., 2003: Characterisation of different pro- venances of late flushing pedunculate oak (Quercus robur L.) with chloro- plast markers. Allg. Forst- und Jagdzeitung 174, 227-231. GAILING, O., WACHTER, H., SCHMITT, H.-P., CURTU, A.-L., FINKELDEY, R., 2007: Characterization of different provenances of Slavonian pedunculate oaks (Quercus robur L.) in Münsterland (Germany) with chloroplast DNA markers: PCR-RFLPs and chloroplast microsatellites. Allg. Forst- und Jagdzeitung 178, 85-90. GEHLE, T., 1999: Genetische Differenzierung der Eiche (Quercus robur) in Nordrhein-Westfalen. Allg. Forst- und Jagdzeitung 170, 183-187. HESMER, H., 1955: Die Späteiche in Westfalen und im Rheinland. Forst- archiv 26, 197-203. KÖNIG, A.O., STAUBER, T., 2004: Haplotypenbestimmung als Hilfsmittel. In: LÖBF/NRW (ed.), Vitalität und genetische Variabilität der Eiche in Nord- rhein-Westfalen. LÖBF/NRW, Recklinghausen. KÖNIG, A.O., ZIEGENHAGEN, B., VAN DAM, B., CSAIKL, U., COART, E., DEGEN, B., BURG, K. et al., 2002: Chloroplast DNA variation of oaks in western Central Europe and genetic consequences of human influences. Forest Ecol. and Manage. 156, 147-166. KREMER, A., KLEINSCHMIT, J., COTTRELL, J., CUNDALL, E.P., DEANS, J.D., DUCOUSSO, A., KÖNIG, A.O. et al., 2002: Is there a correlation between chloroplastic and nuclear divergence, or what are the roles of history and selection on genetic diversity in European oaks? Forest Ecol. Manage. 156, 75-87. LIEPELT, S., SPERISEN, C., DEGUILLOUX, M.-F., PETIT, R., KISSLING, R., SPENCER, M., DE BEAULIUE, J.-L. et al., 2006: Authenticated DNA from ancient wood remains. Ann. Bot. 98, 1107-1111. PETIT, R., AGUINAGALDE, I., BEAULIEU, J., BITTKAU, C., BREWER, S., CHEDDADI, R., ENNOS, R. et al., 2003: Glacial refugia: Hotspots but not melting pots of genetic diversity. Science 300, 1563-1565. PETIT, R., BREWER, S., BORDÁCS, S., BURG, K., CHEDDADI, R., COART, E., COTTRELL, J. et al., 2002a: Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence. Forest Ecol. Manage. 156, 49-74. PETIT, R., CSAIKL, U., BORDÁCS, S., BURG, K., COART, E., COTTRELL, J., VAN DAM, B. et al., 2002b: Chloroplast DNA variation in European white oaks. Phylogeography and patterns of diversity based on data from over 2600 populations. Forest Ecol. Manage. 156, 5-26. PETIT, R., EL MOUSADIK, A., PONS, O., 1998: Identifying populations for conservation on the basis of genetic markers. Conservation Biol. 12, 844- 855. RACHMAYANTI, Y., LEINEMANN, L., GAILING, O., FINKELDEY, R., 2006: Extraction, amplification and characterization of wood DNA from Dipterocarpaceae. Plant Mol. Biol. Reporter 24, 45-55. ROZEN, S., SKALETSKY, H.J., 2000: PRIMER3 on the www for general users and for biologist programmers, In: Krawertz, S., Misener, S. (eds.), Bio- informatic Methods and Protocols: Methods in Molecular Biology, 365- 386, Humana Press, Totowa, NJ, United States. WACHTER, H., 2001: Untersuchungen zum Eichensterben in NRW. Schriften- reihe der Landesforstverwaltung NRW 13, 1-112. WEISING, K., GARDNER, R.C., 1999: A set of conserved PCR primers for the Chloroplast DNA analysis of Slavonian oak stands 171 analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42, 9-19. Address of the authors: Oliver Gailing and Reiner Finkeldey, Institute of Forest Genetics and Forest Tree Breeding, Georg-August University Göttingen, Germany, Büsgen- weg 2, D-37077 Göttingen, email: ogailin@gwdg.de H. Wachter, J. Heyder and H.-P. Schmitt, Landesbetrieb Wald und Holz Nord- rhein-Westfalen, Außenstelle Arnsberg Forstgenbank NRW, Obereimer 2a, D-59821 Arnsberg << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /FRA /ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. The PDF documents can be opened with Acrobat and Reader 5.0 and later.) /JPN /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE /DEU >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [907.087 680.315] >> setpagedevice