Complete mitochondrial DNA sequence of globe fish Tetraodon lineatus (Linnaeus, 1758) and the phylogenetic analysis of tetraodontidae.

Globe fish, Tetraodon lineatus (Linnaeus, 1758) is an ornamental freshwater fish in China. In this study, the complete mitochondrial genome of globe fish was first determined. The entire mitochondrial DNA sequence (mtDNA) sequence was 164595bp in length and consisted of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and a control region (CR). Its mitochondrial genome had the common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures.

The globe fish, like other puffers can inflate when it’s stimulated and carry the tetrodotoxin. It’s a large puffer fish, with yellow stripes, short spines, red or orange eyes (Roberts 1986). However, its natural resources are rare, molecular researches are still less, the mitochondrial genome is not available till today. There are so many questions that we are not clear. Why some puffers can bear TTX, others can’t? To a same TTX-bearing species, the ovary with high quantitative TTX, but the testis without any TTX, why is it so? Who are the TTX-bearing puffer, the oldest ancestors and what the evolution-pattern of puffers is? It will help these researches, when a complete mitochondrial DNA sequence of globe fish is obtained.

In this study, the sample (globe fish) was collected from Luchaogang (30°51′4″, 121°51′26″), Shanghai, China. Pectoral fins were stored in 75 % ethanol at 4 °C. The sample (LCG0010) was kept in the college of Fisheries and Life Science, Shanghai Ocean University. Genomic DNA was extracted using phenol/Chloroform procedure. Partial mitochondrial DNA sequences of globe fish were amplified via PCR using 18 pairs of primers, which were designed according to the experiment. Subsequently, based on received sequences, some additional primers were designed to supplement residual gaps. DNA Baser Sequence Aligner V3.5.4 software (Heracle BioSoft SRL Romania, http://www.DnaBaser.com); (Li & Liu 2014) was used to sequence analysis and assembly. The assembled mitochondrial genome was annotated by MitoFish and Mitoannotator (Iwasaki et al. 2013), then submitted to GenBank. Simultaneously, 23 complete mitochondrial genomes of the Tetraodontidae were downloaded by NCBI. Finally, the phylogenetic analysis was performed using MEGA v.5.1 (http://www.megasoftware.net/index.php) (Tamura et al. 2011), and the number of bootstrap replicates is 1000.

The complete mitochondrial genome of globe fish was 16,459 bp in length (KT715694). It has a typical mitochondrial genome structure, consisted of 22 transfer RNA (tRNA) genes, 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes were encoded on the heavy strand. The length of all tRNAs ranging from 64 to 74 bp and their anti-codons were consistent with other fish of Tetraodontidae. Except for COI with GTG, other 12 protein-coding genes were initiated with the orthodox ATG. They had two types of intact stop codons (TAA and TAG) and two types of incomplete stop codons (TA- and T-).The content A + T(52.57%) was higher than G + C content (47.43%), which was similar to those of Tetraodon mbu (Yamanoue et al. 2011) and Tetraodon miurus (Yamanoue et al. 2011).

The phylogenetic tree was constructed using MEGA5.1 to analyze the phylogenetic relationship of globe fish, and the results of the method were concordant. The tree of nucleotide sequences for all protein-coding genes in Tetraodontidae is shown in Figure 1. It is consistent with the taxonomic status (Igarashi 2013). Teraodon miurus and Teraodon mbu formed the sister group. Teraodon miurus, Teraodon mbu and globe fish constituted the sister branch, which is in accordance with the research of Yoji Igarashi’s (2013). It was suggested that the taxonomic research might lose some significant evolutionary characters.

Figure 1.

Phylogenetic relationship of the Tetraodontidae.