DNA sequencing
Primer pairs LPPGLU (forward) - H414 (reverse) designed to amplify a fragment of the mitochondrial control region (Liukkonen-Anttila et al. , 2002) and L14578 (forward) - H16065 (reverse) of the cytochrome b region (Crowe et al. , 2006), were used. Although successful in the largest proportion of the samples, DNA extraction from feather samples are often of lower DNA quantity and quality. For these samples, primers amplifying smaller sections of these two loci were designed. The longer successful sequences amplified from the blood/tissue samples were aligned to locate conserved regions for the primer design. Primer3 (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) was used to obtain potential primer pairs amplifying a product of roughly 400bp. The primers designed for control region were PER_CR_Frag2F (TGGTTATGCTCGACGTACCA) and PER_CR_Frag2R (ATTCCCCATACACGCAAACC) and those for Cytb were PER_CytB_Frag2F (TGATGAAACTTCGGCTCCCT) and PER_CytB_Frag2R (GGTCGGGTTGTCAACTGAGA).
The total volume of the polymerase chain reaction of the control region was 30 μl in which 100 ng of genomic DNA was amplified, using 1 unit of Qiagen Taq polymerase, 0.2 mM dNTPs, 1 pmol of each primer, 2 mM MgCl2, 3 μl of 10 X Reaction Buffer and 0.3 μl of 100 X BSA. Thermal cycling amplification conditions were as follows: initial denaturation at 94oC for 2 min, followed by 33 cycles of strand denaturation at 94 oC for 1 min, annealing at 54 oC for 1 min and primer extension at 72oC for 1.5 min and a final 5 min elongation time at 72oC.
For cytochrome b, 100 ng of genomic DNA was amplified in 25 μl total volume of polymerase chain reaction, using 1 unit of Qiagen Taq polymerase, 0.2 mM dNTPs, 1 pmol of each primer, 1.5 mM MgCl2, 3 μl of 10 X Reaction Buffer and 0.3 μl of 100 X BSA. Thermal cycling amplification conditions were as follows: initial denaturation at 94 oC for 5 min, followed by 35 cycles of strand denaturation at 94 oC for 1 min, annealing at 52 oC for 1 min and primer extension at 72oC for 2 min and a final 8 min elongation time at 72oC. The PCR products were purified using the Nucleospin Extra kit (Macherey-Nagel, Duren, Germany) and sequenced by Sanger method using an ABI 3730XL DNA Analyzer.
The shorter control region and cytb fragments were run using the same PCR conditions. Reaction volumes were 10 μl, containing 7 μl HotStart PCR master mix (Thermofisher Scientific), 1 μl forward primer (10μM), 1 μl reverse primer (10μM) and 1 μl template DNA at 5ng/μl concentration. Thermal cycling amplification conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of strand denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec and primer extension at 72 °C for 1 min and a final 10 min elongation time at 72 °C.