DNA extraction
, polymerase chain reaction amplification and sequencing
Genomic
DNA
was extracted from spine muscle at the base of the primary spines using the method of Boom et al. (1990). Two gene markers were partially amplified: the 16S rRNA gene using the primers ar-L and br-H ( Palumbi et al. 1991), and the COI gene (as an additional barcode) using the primers LCO1490 and HCO2198 ( Folmer et al. 1994). A 10 µL polymerase chain reaction (PCR) solution was prepared by combining 1 µL of template
DNA
with 9 µL of pre-mixed solution. The pre-mix consisted of 42 µL of deionised water (DW), 10 µL of 10× Ex Taq buffer (TaKaRa, Japan), 8 µL of deoxynucleotide triphosphates, 5 µL each of 10 pmol/µL forward and reverse primers, 0.5 µL of 5 U/µL Ex Taq polymerase, and 20 µL of betaine solution.
Amplification was performed using touch-down PCR on a TaKaRa Thermal Cycler Dice Gradient with the following cycling conditions. For 16S: 98°C for 1 min; 10 cycles at [98°C for 10s, 50°C (decreasing by 1°C for each cycle) for 30s, and 72°C for 45s]; 25 cycles at [98°C for 10s, 40°C for 30s, and 72°C for 45s]; ending with 72°C for 7 min. For COI: 98°C for 1 min; 10 cycles at [98°C for 10s, 45°C (decreasing by 1°C for each cycle) for 30s, and 72°C for 45s]; 25 cycles at [98°C for 10s, 35°C for 30s, and 72°C for 45s]; ending with 72°C for 7 min.
The amplified product was mixed with a loading buffer with SYBR Green dye (TaKaRa, Japan) and visualised via electrophoresis on a solidified 1% agarose gel. The gel electrophoresis confirmed the presence of the expected amplification products. The PCR products were subsequently purified using the ExoSAP protocol, involving the addition of 28 mU of Exonuclease I (TaKaRa, Japan) and 6 mU of Shrimp Alkaline Phosphatase (TaKaRa, Japan) in an 18 µL reaction volume. This mixture was incubated for 30 min at 37°C to degrade residual primers and nucleotides, followed by enzyme deactivation at 80° C for 15 min. The purified PCR products were subjected to cycle sequencing using the BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, USA) with a reaction volume of 5 µL. The sequencing reaction consisted of 1 µL of 0.8 pmol/µL of primer (the same primers used for PCR), 2 µL of the purified product, 0.5 µL of of 5× Sequencing Buffer, 0.5 µL of DW, and 1 µL of BigDye Terminator v. 3.1 Ready Reaction Mix. The BigDyereaction conditions were as follows: 25 cycles of [96°C for 10s, 50°C for 0.5 s, and 60°C for 4 min]. Purification and BigDye reactions were performed in a 2720 thermal cycler (Applied Biosystems). After the BigDye reaction, the products were purified by precipitating the
DNA
using sodium acetate and 75% ethanol, followed by centrifugation to form
DNA
pellets. These pellets were then dissolved in 14 µL of formamide in preparation for gene sequencing. The gene markers were sequenced using a 3130 Genetic Analyser (Applied Biosystems, USA) with the 3130KB_POP&_V3 module. Base calling was performed using GeneStudio Pro v. 2.2.0.0 (GeneStudio Inc., GA, USA). To verify the quality and accuracy of the sequences, a BLAST search ( Altschul et al. 1997) was conducted on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) to check for potential contamination or misidentification.
