Chloroplast DNA Analysis in Oak Stands
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by JKI Open Journal Systems (Julius Kühn-Institut) 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 can be recognized at cpDNA markers (DUMOLIN-LAPÈGUE et al., Slavonian oaks (Quercus robur subsp. slavonica) have been intro- 1998). duced into Germany in the second half of the 19th century from the An European-wide inventory of more than 2600 populations of white lowlands of the rivers Save and Drava in today’s Croatia. If compared oaks using PCR-RFLPs of four non-coding chloroplast regions (PETIT to indigenous oak stands, they are characterized by good growth, et al., 2002b) revealed a total of 45 haplotypes with a characteristic comparatively low seed production and a late bud burst. Based on distribution in Europe (PETIT et al., 2002b). the information of European-wide variation patterns at chloroplast In a former study we adapted chloroplast microsatellites (DEGUIL- DNA markers in oaks we adapted chloroplast microsatellites for LOUX et al., 2003; WEISING and GARDNER, 1999) for the analysis of the analysis of all oak stands of presumably Slavonian origin in the oak stands in the Münsterland region. By applying these markers Münsterland and lower Rhine regions. We were able to distinguish we were able to distinguish most chloroplast haplotypes and found between Slavonian haplotypes with no natural occurrence in the study complete congruence with the PCR-RFLP procedure (GAILING area and indigenous types that do not occur in the Balkan region. A et al., 2007). These and an additional new marker were used in the generally high differentiation among stands was observed at chloro- present study in order to determine the haplotype composition of all putative Slavonian oak stands in North Rhine-Westphalia (Germany). plast markers (GST = 0.674). Based on the haplotype information and historic records we found that stands with Slavonian material Three control stands were included in the analysis that have been have been established between the years 1878 and 1903. In a total of identified to be indigenous according to their growth habit, bud 910 analysed trees the Slavonian haplotypes 5, 2 or 17 were the most phenology and /or due to the early stand establishment before the frequent ones but a considerable amount of samples with indigenous introduction of Slavonian oaks into Germany. Here, we present the haplotype 1 or haplotype10 with presumed origin in Southwestern results of the cpDNA analysis of 33 newly investigated stands. Haplo- Europe was also present. A clear association between haplotype 2 type frequency and diversity within and among stands were analysed and late bud burst was detected in adult stands and in a field trial by including data from 17 formerly analysed stands (GAILING et al., established with seeds from Slavonian and indigenous oak stands. 2003; GAILING et al., 2007). The information about the haplotype composition in all Slavonian The aims of the present study are to give an overview of the haplotype stands can be used as reference for the certification of reproductive composition of all pedunculate oak stands with presumably Slavonian material. The analysis of cpDNA haploytpes in old oak stands that origin in North Rhine-Westphalia and to determine the time frame had been established before the introduction of foreign seed material in which stands with different origins (haplotypes) have been estab- can give valuable information for the identification of indigenous lished. More specific aims are the development of reliable and easy- oak stands. 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 Introduction (identified by the chloroplast haplotype) and the putatively adaptive Slavonian pedunculate oaks had been introduced into Germany character bud burst. (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 Materials and methods indicated that Slavonian oaks originated from the lowlands of the Plant material rivers Save and Drava in today’s Croatia (GAILING et al., 2007; GEHLE, 1999; HESMER, 1955; WACHTER, 2001). Slavonian oaks in Germany A total of 50 stands (mean of 18.2 samples per population) of Quercus are characterized by late bud burst, fast growth and long clear boles robur in the Münsterland and from the Lower Rhine region have when compared to indigenous oaks that had been planted at the same been studied including all putative stands of Slavonian oak (Quercus time in the same stand (GAILING et al., 2003; WACHTER, 2001). robur ssp. slavonica) in North Rhine-Westphalia (Tab. 4). Most of Earlier studies indicated that stands established with Slavonian oaks them have been characterized as Slavonian oaks according to their flushed two or three weeks later than neighboring indigenous oak growth habit, bud phenology (late flushing) and according to his- stands, possibly resulting in better resistance to late frosts and lower torical documents. Samples from one stand (Croatia) were collected susceptibility to pests (e.g. feeding damage by the European oak directly in Croatia (Vinkovsi). Three of the stands were characterized leaf roller Tortrix viridana) (GAILING et al., 2003; WACHTER, 2001). to be indigenous Q. robur according to growth habit and historic However, the number of presumably Slavonian stands that have been documents and were used as references. Seventeen of the stands were characterized at chloroplast DNA markers and for bud phenology included in earlier studies (GAILING et al., 2003; GAILING et al., 2007). was rather limited. Additionally, four provenances of presumably Slavonian origin grown Chloroplast DNA (cpDNA) markers can give valuable information in a field trial in Dormagen (Abt. 33C/F, Frh. v. Nagel-Doornick; about the geographic origin of oaks and other angiosperms. The Abt. 35C1; Abt. 161 A1, Frh. v. Boeselager; Abt. 343a3, FA Peine) chloroplast genome is uniparentally inherited in maternal lineages; and a reference stand (Frh. v. Vittinghoff-Schell) were characterized usually, recombination does not occur. Thus, patterns of seed dispersal 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 cpSSRs. However, they were easily distinguishable by amplification grown from seed material that was collected in stands in 1988. of the trnD-trnT region and restriction with TaqI. Differences in the restriction pattern were clearly recognized on a 2% agarose gel. DNA isolation Total genomic DNA was extracted from fresh leaves (a small slice Data analysis of about 1 cm2) or from buds with the DNeasy Plant Kit for 96 probes As in most angiosperms the chloroplast genome in oaks is maternally from Qiagen (Hilden, Germany). The amount of DNA was checked inherited reflecting the dispersal by seeds (DUMOLIN-LAPÈGUE et al., on 1% agarose gels after staining with ethidium bromide. 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 PCR-RFLP technique markers (see Tab. 2). Absolute haplotype frequencies were determined The following non-coding regions of the chloroplast genome trnD- in each stand. Haplotype frequencies were used as input to calculate trnT with TaqI (DT), trnC-trnD with TaqI (CD), psa-trnS with HinfI within-population diversity (HS) and total diversity of haplotypes (HT) (AS) were studied with the PCR-RFLP technique (DEMESURE et al., in the program RAREFAC (PETIT et al., 1998). Likewise, allelic rich- 1995). The PCR profile was as described by DEMESURE et al. (1995). ness as a measure adjusting for different sample sizes was calculated PCR amplification was performed in a 15 µl volume containing about after rarefaction in the program RAREFAC (PETIT et al., 1998) with 10 ng template DNA, 1x Qiagen PCR buffer; 1x Q-solution (Qiagen), a rarefraction size equalling the smallest sample size. GST ((HT-HS)/ HT) was calculated for the partitioning of diversity among stands. 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- RST was also calculated as a similar measure of population differen- actions were performed by adding 5 µl of PCR product to a mix tiation that is derived from estimates of allelic richness (PETIT et al., 1998). 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 Assessment of bud burst 8% polyacrylamid (PAA) gel, stained with SYBR Gold (Molecular probes) and visualized under UV light. The interpretation of the re- Bud burst was scored on a scale ranging form 0 (buds closed) to 5 striction pattern followed PETIT et al. (2002b). Control probes were (leaves fully expanded). When samples were collected in the mid of used to assign the resulting patterns to haplotypes described in the May 2006, stands with extreme phenotypic values for bud burst were European-wide inventory of oak species at cpDNA markers.