2.2 Morphological identification
The body length, weight and other measurable traits of the specimen were
measured. The morphological taxonomic characteristic of
Tetraodontiformes is mainly base on the body shape, fin number, stripes,
spots, body color and mouth shape, etc described by Chen & Zhang (2015)
and Su & Li (2002).
2.3 DNA extraction and quality
inspection
DNA was extracted from the muscle of fish by using TIANamp Marine
Animals DNA Kit (TIANGEN BIOTECH (BEIJING) CO.,LTD.). For detailed steps
of DNA extraction, refer to the kit manual. DNA samples were stored at
-20 ℃.
Nano-300 micro-spectrophotometer (Allsheng) was used to detect the
concentration and purity of DNA samples, and the quality of the DNA
samples was detected by 1% agarose gel.
2.4 PCR amplification and DNA
sequencing
The published universal primers for fish DNA barcodes (Ward et al.,
2005) were used for PCR amplification and sequencing, and the length of
the amplification product was 707bp. The primers were as follows:
COIF: 5’-TCAACCAACCACAAAGACATTGGCAC-3’
COIR: 5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’
PCR amplification was in total volume of 25 μL, containing 12.5 μL Taq
Mixture (2ⅹ), 2 μL DNA template, 1 μL of each primer (10 μM) and 8.5 μL
ddH2O. Thermo cycling were carried out as follows: 94 ℃ for 5 minutes,
94 ℃ for 30 seconds, 53~56 ℃ for 30 seconds, 72 ℃ for 1
minute, cycle 35 times from step 2 to step 4, finally 72 ℃ for 10
minutes. PCR products were detected by 1% agarose gel, and the
qualified samples were sent to BGI in Qingdao for direct bidirectional
sequencing.
2.5 Data analysis
The raw sequences were assembled by seqman of Lasergene software package
(Swindell & Plasterer, 1997; Burland, 1999) and aligned by Cluster W in
BioEdit version 7.0.9 (Hall, 1999). After correction and alignment, the
effective sequence length was 687 bp. The haplotypes were analyzed by
DNASP version 5.10.01 (Librado & Rozas, 2009). Each haplotype sequence
was compared for similarity in NCBI (https://www.ncbi.nlm.nih.gov/) and
BOLD
(http://www.barcodinglife.org/).
The top matches showing ≥ 98% similarity were used as the preliminary
identification results. Then based on Kimura- 2- parameter (K2P) mode
(Kimura, 1980), the genetic distance of different taxonomic levels of
Tetraodontiformes was calculated.
Bothus myriaster and Glyptocephalus stelleri were selected
as the outgroups, based on the haplotypes of COI gene of
Tetraodontiformes, the Neighbor-joining tree (NJ tree) of
Tetraodontiformes was constructed in Mega 7.0 (Kumar, 2015).