Cytochrome oxidase subunit I gene fragment amplification and
sequencing
A fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene
was amplified for a representative subset of 86 individuals using the
FishF1 (5ˈ-244 TCAACCAACCACAAAGACATTGGCAC-3ˈ) and FishR1
(5ˈ-TAGACTTCTGGGTGGCCAAAGAATCA-3ˈ) primers (Ward et al. 2005) in a total
volume of 20 μl with 0.2 μl of Dream Taq Polymerase (Thermo Fisher
Scientific), 2 μl of Dream Taq Buffer 10X (Thermo Fisher Scientific),
0.4 μl of each primer and 50 ng of total DNA using the following
profile: an initial denaturation step at 95°C for 3 min, 35 cycles of 30
sec at 98°C, 30 sec at 54°C and 60 sec at 72°C, and a final extension of
72°C for 10 min. Products were visualized on 1.7% agarose gels,
purified with GE Healthcare Illustra ExoProStar™ (ref. US77705) and
Sanger sequenced. The newly generated 86 sequences were edited using
SeqTrace 0.9.0, submitted to Genbank (Accession numbers
MT037084-MT037149, MT037151-MT037170) and aligned with BioEdit (v7.2.5)
together with other publicly available representative COI sequences of
albacore (accession number KT074102) and ABFT (accession number
DQ107585), including the alalunga-like (accession number GQ414567)
haplotypes (Table S2). Diagnostic positions between ABFT and albacore
haplotypes were used to detect mitochondrial introgression from albacore
to ABFT samples.