sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ Faculty of Forestry Journal
Minisatellite sequences are repeated over the genome and exist in most organisms
providing an obtainable and highly variable probe and PCR amplification (Karlsson,
1993; Högberg et al., 1995). In most cases they have been used to study variation
among populations.
In our study, we used M13 minisatellite core sequence as a primer in PCR based
DNA fingerprinting technique to study level of genetic variation among G. fuscum
populations.
2. MATERIAL AND METHODS
Telial horns were collected from trunk lesions in Sütçüler, Bucak-Aziziye and
Beşkonak sites. In Sütçüler three stands 3-8 km apart were sampled. The stands in
Beşkonak and Bucak-Aziziye were 40 and 70 km west from the Sütçüler stands,
respectively.
In each stand telia horns were sampled from ten trees, placed into plastic bags,
and stored in the laboratory at -20 ºC until DNA extraction.
Figure 1. Locations of the sampled stands (crosses).
2.1. DNA extraction and PCR amplification
Telia were grounded using liquid nitrogen and DNA was extracted with Qiagen
DNeasy Plant Mini Kit. PCR amplification profiles were obtained using M13
minisatellite core sequence as a primer. Reactions were performed in volumes of
50 μl containing Tris-HCl 10 mM, pH 8; dNTPs 0.2 mM; MgSO4 2 mM; Tsg
polymerase 1,25 U; primer (M13) 2 μM; approximately 10 ng of template DNA.
PCR was conducted using a hot start step at 95°C for 10 min, followed by 45
cycles at 95°C for 1 min, 48°C for 30 s, 72°C for 2 min and a final extension at
72°C for 10 min.
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