3.2. Molecular methods

Total DNA was isolated from blood, muscle, liver or kidney with a standard phenol-chloroform extraction (Sambrook 1989) or from feathers with a Chelex-based method (Walsh et al. 1991).

The complete mitochondrial control region and the flanking areas (in total 1227 bp) were amplified and sequenced to study the phylogenetic relationships of the Anser species (II). To study the population genetic structure and phylogeography of the lesser white-fronted goose (III) and the genetic composition of the captive lesser white-fronted goose population (IV), the hypervariable 5’ fragment of the control region was sequenced. In study II, this 221 bp fragment was shown to be the most variable part of the control region among geese, and it contains approximately half of the variable nucleotide positions in the complete control region. PCR was used to amplify the mitochondrial fragments with specific primers (primer sequences presented in the original papers, see also 3.3. for discussion regarding the specificity of primers).

PCR amplification products were purified from 1% agarose gel (Glenn & Glenn 1994) to remove excess primers and dNTPs. All sequencing was done using double-stranded PCR products directly as templates to detect possible heteroplasmy and to diminish the problems stemming from errors made by DNA polymerase during the PCR. At first, the sequencing was done manually (Bernatchez et al. 1992). Later on, sequencing was performed with the ABI 377 DNA Sequencer according to manufacturer’s instructions.