Pityogenes chalcographus is a widely distributed spruce pest in Eurasia (KNIZEK et al. 2005). In 70ies, E. Führer studied the intraspecific variation of this spruce bark beetle and detected race differentiation among European populations based on crossing experiments (FÜHRER 1977), morphological characters FÜHRER 1978) and allozyme electrophoresis (RITZENGRUBER 1990). In order to verify the hypothesis differentiation, we analysed diverse European P. chalcographus populations using the Cytochrome Oxidase gene (COI) of the mitochondrial DNA. The complete COI gene of 96 individuals was sequenced. In facilitate the screening of the European populations, we applied a PCR-SSCP method. This polyacrylamide electrophoresis technique offers a sensitive but inexpensive, rapid and convenient method for detecting polymorphisms, reducing the amount of samples that require sequencing (SUNNUCKS et al. 2000).Der Kupferstecher Pityogenes chalcographus (Coleoptera, Scolytidae) gehört zu den am weitesten verbreiteten Borkenkäfern Europas. Käferpopulationen aus verschiedenen Gebieten Europas zeigten eine hohe Divergenz hinsichtlich der Nukleotidzusammensetzung des mitochondrialen Cytochrom Oxidase I Gens. Die Unterschiede betragen bis zu 2,26% zwischen einzelnen Herkünften. Die phylogenetische Auswertung der Sequenzen des kompletten Gens (1503bp) ergab einen 6-astigen Baum mit insgesamt 34 Haplotypen. Ausgehend von dieser Struktur wurden Primerpaare entworfen, um jeweils etwa 250 bp lange mutationsreiche Abschnitte des COI Gens zu amplifizieren. Die PCR Produkte wurden mittels Single Strand Conformation Polymorphism (SSCP) untersucht. Zwischen den einzelnen Haplotypen konnten zahlreiche Polymorphismen erkannt werden. Diese eignen sich als schnelles Werkzeug zum screening einzelner Individuen und verringern signifikant die Notwendigkeit klassischer Sequenzierreaktionen

Arthofer, Wolfgang

Avtzis, Dimitrios N.

Stauffer, Christian

English

Hochschulschriftenserver - Universität Frankfurt am Main

MITT. DTSCH. GES. ALLG. ANGEW. ENT. 15 GIESSEN 2006    173Identifying haplotypes of Pityogenes chalcographus (Col., Scolytidae) by Single Strand Conformation Polymorphism (SSCP)   Dimitrios N. Avtzis, Wolfgang Arthofer & Christian Stauffer  Institut für Forstentomologie, Forstpathologie & Forstschutz der Boku Wien  Zusammenfassung:  Identifizierung von Haplotypen bei Pityogenes chalcographus (Col., Scolytidae) durch SSCP. Der Kupferstecher Pityogenes chalcographus (Coleoptera, Scolytidae) gehört zu den am weitesten verbreiteten Borkenkäfern Europas. Käferpopulationen aus verschiedenen Gebieten Europas zeigten eine hohe Divergenz hinsichtlich der Nukleotidzusammensetzung des mitochondrialen Cytochrom Oxidase I Gens. Die Unterschiede betragen bis zu 2,26% zwischen einzelnen Herkünften. Die phylogenetische Auswertung der Sequenzen des kompletten Gens (1503bp) ergab einen 6-astigen Baum mit insgesamt 34 Haplotypen. Ausgehend von dieser Struktur wurden Primerpaare entworfen, um jeweils etwa 250 bp lange mutationsreiche Abschnitte des COI Gens zu amplifizieren. Die PCR Produkte wurden mittels Single Strand Conformation Polymorphism (SSCP) untersucht. Zwischen den einzelnen Haplotypen konnten zahlreiche Polymorphismen erkannt werden. Diese eignen sich als schnelles Werkzeug zum screening einzelner Individuen und verringern signifikant die Notwendigkeit klassischer Sequenzierreaktionen.   Key words: Scolytidae, Pityogenes, SSCP, phylogeography, mitochondrial DNA, PAGE  Dimitrios N. Avtzis, Wolfgang Arthofer & Prof. Dr. Christian Stauffer, Department für Wald- & Bodenwissenschaften, Universität für Bodenkultur, 1190 Wien.    E-mail: stauffer@ento.boku.ac.at; DNA & WA contributed equally to that paper.   Pityogenes chalcographus is a widely distributed spruce pest in Eurasia (KNIZEK et al. 2005). In the mid 70ies, E. Führer studied the intraspecific variation of this spruce bark beetle and detected race differentiation among European populations based on crossing experiments (FÜHRER 1977), morphological characters (FÜHRER 1978) and allozyme electrophoresis (RITZENGRUBER 1990). In order to verify the hypothesis of race differentiation, we analysed diverse European P. chalcographus populations using the Cytochrome Oxidase I gene (COI) of the mitochondrial DNA. The complete COI gene of 96 individuals was sequenced. In order to facilitate the screening of the European populations, we applied a PCR-SSCP method. This polyacrylamide electrophoresis technique offers a sensitive but inexpensive, rapid and convenient method for detecting DNA polymorphisms, reducing the amount of samples that require sequencing (SUNNUCKS et al. 2000).  Materials and Methods Beetles were collected from several European locations given in table 1. They were directly put into absolute ethanol and stored there until use. Based on the sequences and mutational site distributions of 96 previously examined individuals of P. chalcographus several forward and reverse primers located in conserved DNA stretches along the COI gene were designed. Focus was laid to amplicon sizes between 200 and 260bp and annealing temperatures close to 60°C allowing the use of different primer combinations in the same cycling procedure. Finally two primer systems PC6 and PC4 (Table 2) were selected for subsequent investigations. PCR was performed in 10µl reactions containing 0.2µM forward and reverse primer, 50µM of each dNTP, 0.8µl template DNA, 0.4U Taq DNA polymerase (Sigma) and the PCR buffer provided by the manufacturer. Cycling conditions on a PTC-100 thermocycler (MJ Reseach) comprehend an initial denaturation at 94°C for 3min followed by 33 cycles of 94°C for 40sec, 60°C for 40sec and 72°C for 1min. Final elongation time was 15min at72°C. MITT. DTSCH. GES. ALLG. ANGEW. ENT. 15 GIESSEN 2006    174  ht2 ht34 ht1 ht3 ht8 ht5 ht4 ht7 ht6 ht9 ht11 ht10 ht20 ht21 ht19 ht18 ht12 ht13 ht14 ht15 ht16 ht17 ht22 ht32 ht30 ht23 ht28 ht31 ht29 ht33 ht27 ht26 ht25 ht24928180958570710.002    Fig. 1:   Phylogenetic relationships of the 34 haplotypes of P. chalcographus calculated by NJ algorithm and based on the Tamura-Nei (TAMURA & NEI 1993) substitution model. Pityogenes trepanatus and Pityogenes hopkinski (AF:113395) were taken as outgroups.  PCR products were mixed with 1 vol of denaturing loading dye (96% formamide, 0.1% bromophenole blue, 0.1% xylene cyanol), incubated 5min at 95°C and immediately put on ice. 8µl of the mixture were loaded onto a gel containing 0.5x TBE, 5% glycerol, 10% acrylamide, 1% Bis and 0.2% of TEMED and APS. Electrophoresis was performed in a 200mm dual slab gel unit (C.B.S. Scientific) at 250V for 20 hrs. A constant gel temperature of 4°C for PC6 respectively 15°C for PC4 was provided by a circulating water flow. Gels were stained with 1x SYBR–Gold® (Molecular Probes) in 0.5x TBE for 15 min and visualized on a transilluminator.  A phylogenetic analysis was calculated using the Mega2 software (KUMAR et al. 2001) applying Neighbor Joining with the Tamura – Nei substitution model (TAMURA & NEI 1993).  MITT. DTSCH. GES. ALLG. ANGEW. ENT. 15 GIESSEN 2006    175Table 1: Information on the populations of P. chalcographus.  Collection site  Country  Host    Lon.  Lat. Korgen Norway  Picea abies 66°10 N  13°40 E Eina Norway  Picea abies 60°38 N  10°36 E Kangashäkki Finland  Picea abies 62°36 N  25°44 E Rolfstorp Sweden  Picea abies 57°06 N  12°17 E Vilnius Lithuania  Picea abies 54°41 N  25°19 E Hajnowka Poland  Picea abies 52°45 N  23°36 E Bistra Romania  Picea abies 46°30 N  23°10 E Tharandt Germany  Picea abies 50°59 N  13°35 E Hofoldinger Forst  Germany  Picea abies 48°03 N  11°35 E Zwardon Slovakia  Picea abies 49°28 N  18°50 E Banska Stiavnica  Slovakia  Picea abies 48°28 N  18°56 E Uhlirske Janoviee   Czech Rep. Picea abies 49°50 N  15°10 E Bielersee Swiss  Chalcoprax 47°10 N    7°20 E Prinzensdorf Austria  Picea abies 48°11 N  15°35 E Kalkalpen Austria  Picea abies 47°53 N  14°15E Kärnten Austria  Picea abies 46°37 N  14°37 E Brixen   Italy  Picea abies 46°43 N  11°39 E Tolmezzo Italy  Picea abies 46°24 N  13°01 E Asiago Italy  Picea abies 45°52 N  11°30 E Drama Greece  Chalcoprax 41°09 N  24°08 E   Results and Discussion COI revealed six significant clades with a maximum nucleotide divergence of 2,26% (Fig. 1). With the use of PC6 we determined three SSCP profiles that represent haplotypes of the clades I, IIa and IVa (Fig. 2a), while the application of PC4 enabled us to distinguish two more SSCP profiles (Fig. 2b) that link to haplotypes within clades IVb and IVc. A subsequent combination of the primer systems PC6 and PC4 (Table 2) achieved a sufficient resolution to assign any unknown sample directly to 5 out of 6 clades. Samples that fail to affiliate with any of these five clades, fall consequently into clade IIIb.   Table 2:  Primers used in the SSCP analysis  Primer System  Sequence/F  Tm/F  Sequence/R  Tm/R size of product (bp) Pc 4 tcc aga tgc cta cct tct ctg  55,5 tcc aat gca cta atc tgc cat att a  53,8 252 Pc 6 gcc cca gat ata gca ttt cc  53,2  aat tcc tga tat atg aag gct g  49,9  200  Based on the SSCP profiles, we proceeded to the screening of populations from 20 different localities (Table 1). We analysed about 16 individuals per population. Thus out of about 322 individuals that were screened, 37% were determined after the application of PC6. These 117 samples are assigned to clades I (45,3%) and IVa (54,7%), while the gel phenotype of clade IIa was not found in any electrophoresis. The subsequent application of PC4 on the 63% of the samples that remain unidentified is required in order to determine their affiliation to the clades. Even though, sequencing has become less expensive it remains a limiting factor in screening lots of individuals. In the analysis of P. chalcographus, the identification of 34 haplotypes encouraged us to turn to SSCP. Although this technique has been widely applied in biomedical research, only few studies have been published in entomological research (MARQUEZ  &  KRASFUR  2003). For the screening of European P. MITT. DTSCH. GES. ALLG. ANGEW. ENT. 15 GIESSEN 2006    176 chalcographus populations, this technique proved to be fast and highly sensitive to detect all the haplotypes within the single clades. Thus we will apply this technique now to detect the genetic basis of race differentiation within that species.                a)                                                                              b)                 Fig. 2: a) Gel phenotypes of six individuals after PCR with primer system PC6. b) Gel phenotypes of four individuals after PCR with primer system PC4. Gels were stained with SYBR–Gold®.   Acknowledgements We would like to thank the Austrian Science Foundation (FWF) for funding this project.  References FÜHRER, E. (1977): Studien über intraspezifische Inkompatibilität bei Pityogenes chalcographus. – J. Appl. Ent. 83: 286-297. FÜHRER,  E. (1978): Rassendifferenzierung bei Pityogenes chalcographus L. (Coleoptera,Scolytidae) 1. Morphologische Merkmale. – J. Appl. Ent. 86: 392-402. KNIZEK, M., STAUFFER, C., AVTZIS D., WEGENSTEINER R. (2005): Pityogenes chalcographus – In: Forestry Compendium 2005 CAB International.  KUMAR S., TAMURA, K., JAKOBSEN, I.B., NEI, M. (2001): MEGA2: molecular evolutionary genetics analysis software. – Bioinformatics 17: 1244-1245. MARQUEZ, J.G., KRASFUR, E.S. (2003): Mitochondrial diversity evaluated by the single strand conformation polymorphism method in African and North American house flies (Musca domestica L.). – Insect Mol. Biol. 12: 99-106. ORTÍ, G., HARE, M.P., AVISE, J.C. (1997): Detection and isolation of nuclear haplotypes by PCR-SSCP. – Mol. Ecol. 6: 575-580. RITZENGRUBER, O. (1990): Isoenzymanalyse verschiedener Populationen von Pityogenes chalcographus L. 2. Populationsstruktur, Populationsdifferenzierung. – J. Appl. Ent. 109: 55-63. SUNNUCKS, P., WILSON, A.C.C., BEHEREGARAY, L.B., ZENGER, K., FRENCH, J., TAYLOR, A.C. (2000): SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. – Mol. Ecol. 9: 1699-1710.  TAMURA, K., NEI, M. (1993): Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. – Mol. Biol. Evol 10: 512-526.     Clade I CladeIVa CladeIIa CladeIVb Clade IVc 

Detection and isolation of nuclear haplotypes by PCR-SSCP.

Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.

Isoenzymanalyse verschiedener Populationen von Pityogenes chalcographus L.

MEGA2: molecular evolutionary genetics analysis software.

Mitochondrial diversity evaluated by the single strand conformation polymorphism method in African and North American house flies (Musca domestica L.).

Pityogenes chalcographus – In: Forestry Compendium

Rassendifferenzierung bei Pityogenes chalcographus L. (Coleoptera,Scolytidae) 1. Morphologische Merkmale.

SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology.

Studien über intraspezifische Inkompatibilität bei Pityogenes chalcographus.

Identifizierung von Haplotypen bei Pityogenes chalcographus (Col., Scolytidae) durch SSCP

Identifizierung von Haplotypen bei Pityogenes chalcographus (Col., Scolytidae) durch SSCP

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