98 Y. M. Hokama, D. C. Savi, B. Assad et al.
Figure 1. Map showing the Pantanal colored in gray and the collection site is in the
magnification box, with the points at Santa Emilia Farm illustrating the nine sampled
trees. The collection points were identified by letters and numbers:
1(19.501944ºS/55.601389ºW), 2(19.500833ºS/55.600556ºW),
3(19.500556ºS/55.600278ºW), 4(19.501111ºS/55.600556ºW),
A(19.508548ºS/55.628775ºW), B(19.506286°S/55.610172ºW),
C(19.509930ºS/55.627039ºW), D(19.509038ºS/55.626354ºW) and
E(19.507243ºS/55.615815ºW).
Amplification of the internal transcribed spacer (ITS) region was
performed under the conditions described by White Jr. and Morrow (1990)
with the primers V9G (De Hoog et al. 1998) and ITS4 (White and Morrow
1990). The reactions were performed in an Eppendorf MasterCycler Gradient
Thermal Cycler. PCR amplification products were visualized and quantified
by electrophoresis in a 1.5% agarose gel (w/v), stained with GelRed Nucleic
Acid Gel Stain (Biotium Inc., Hayward, US) and using a Ludwig Biotec
(Ludwig Biotec, Alvorada, BR) molecular weight marker. PCR products were
purified by ethanol precipitation (Fredricks et al. 2005) and directly sequenced
using a DYEnamic ET Dye Terminator Kit (Amersham Biosciences),
following instructions from the manufacturer. The sequencing reactions were
purified using Sephadex gel G-50 column (GE Healthcare) in ELISA wells,