Amplification and Sequencing of VGSC and nad4 Genes

The primers used to amplify exons 21 and 31 of the VGSC gene, which encode for domain II subunit 5 and domain III subunit 6, respectively [31 (link)], as well as the mitochondrial nad4 gene [35 (link)] are described in Additional file 1: Table S2. Polymerase chain reaction (PCR) was carried out in a total volume of 25 µl using standard protocols with 1× reaction buffer, 200 µM dNTP, 0.5 µM forward primer, 0.5 µM reverse primer, 0.02 U/µl DNA polymerase, 17.5 µl water and 1.5 µl DNA sample (1–5 ng). The PCR thermocycler profile consisted of: 1 cycle at 98 °C for 30 s, 30 cycles at 98 °C for 10 s, 56 °C (VGSC gene) or 59 °C (nad4 gene) for 30 s, 72 °C for 30 s and a final cycle at 72 °C for 2 min. PCR products (10 µl each) were detected by 1% agarose gel electrophoresis in TAE buffer, stained using GelRed (Cambridge Bioscience, Cambridge, UK). The samples were sequenced by capillary sequencing. The resulting DNA sequences have been submitted to GenBank under the accession numbers MT721877-MT721961.