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2012) across 3 countries (Croatia, Slovenia and Italy; Figure 1 and Table 1). Specimens were immediately put into eppendorfs with pure ethanol (100 % EtOH) so that DNA would remain intact. DNA extraction was carried out with GenElute Kit (Sigma), following the protocol of the manufacturer. Amplification of an approximately 700bp locus from the mitochondrial Cytochrome Oxidase I (Cox1) gene was carried out with primers LCO2198 (5’-ggtc aac aaa tca taa aga tat tgg-3’) and HCOI1490 (5’-taa act tca ggg tga cca aaa aat-3’), designed for the analysis of D. kuriphilus by Ács et al. (2007). Reactions were 25μl in volume, containing 0.2μl of MyTaqTM (BioLine, GmBH, Germany), 5μl of the provided 5x MyTaqTM Red Reaction Buffer (BioLine, GmBH, Germany), 0.75 μl of each primer (20μΜ), 8 μl of DNA extract and ddH2O to the volume of 25μl. PCR conditions included an initial denaturation step at 94 °C for 3 minutes, followed by 40 cycles at 94 °C for 30 s, 48 °C for 30 s and 72 °C for 90s with a final extension step that lasted 7 minutes at 72 °C. PCR products were then purified with PureLinkTM PCR Purification Kit (Invitrogen) and sent for sequencing with both primers at a commercially available service (Chemia SA, Larissa – Greece). Sequences were initially visualized by ChromasLite® (Technelysium Pty Ltd.) and aligned manually by eye. The haplotype retrieved was deposited in NCBI Genebank under the Ascession Number (KF308606). ClustalX (Thomson et al., 1997) was used to create an alignment of the haplotype with reference sequences acquired from NCBI GeneBank with Accession Numbers (JF411594, JF411595, JF411596, JF411597, JF411598 and DQ286810). Nucleotide diversity and pairwise nucleotide differences were calculated with MEGA version 5 (Tamura et al. 2011).
A polymorphic locus of about 700bp in length from mtDNA’s Cytochrome Oxidase One (Cox1) gene was amplified from 96 individuals. Alignment of a 623bp long locus revealed that every individual exhibits the same mitochondrial haplotype. Careful examination of the sequences together with comparison of the two loci obtained by both primers, verified the occurrence of only one haplotype in all populations from Italy, Slovenia and Croatia. When this unique European haplotype was further aligned with the only reference sequences of D. kuriphilus deposited in NCBI Genebank, it matched perfectly with the Italian haplotype under the Accession Number DQ286810 (Ács et al. 2007).