Complete mitochondrial genome of Opisthotropis andersonii (Serpentes: Colubridae) determined using next-generation sequencing

Abstract

The complete mitochondrial genome (mitogenome) of Opisthotropis andersonii was first determined using illumina next-generation sequencing. The total length of genome was 18,297bp with a slightly AT bias (59.57%), and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, 2 control regions, and an origin of light-strand replication. All genes are distributed on the H-strand, except ND6 gene. A phylogenetic tree was constructed based on the 13 PCG sequences of O. andersonii and other related snakes to analyze their phylogenic relationship, exhibiting the closest relationship with Thermophis baileyi and Nerodia sipedon.

Keywords:

• Mitochondrial genome
• Opisthotropis andersonii
• phylogenic relationship

Opisthotropis andersonii was listed as a near threatened species in the Red List (IUCN 2018) and was considered non-venomous, belonging to the genus Opisthotropis of family Colubridae, which was distributed in China and Vietnam (Zhao et al. 1998; David et al. 2011). There are 21 species in the genus Opisthotropis presently (Wang et al. 2017). But, the complete mitochondrial genome of these species is not published and few molecular data are available. In this paper, we determined and described the complete mitogenome of O. andersonii in order to obtain basic genetic information about this species, enrich the Opisthotropis species genome resource, and promote the further research concerning the related species.

Opisthotropis andersonii was collected from the Baiyun mount, Guangzhou, China. It was stored in the Specimen Museum of Guangdong Institute of Applied Biological Resources, GDAS. Whole genomic DNA was extracted from the specimen’s liver using the DNeasy$\mathrm{R}$ Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Genomic DNA was prepared in 150 bp paired-end libraries, tagged and subjected to the high-throughput Illumina Hiseq Xten platform, and yielded 29,750,352 raw reads. DNA sequences were assembled using MITObim version1.9 (Hahn et al. 2013). Comparing with the Nerodia sipedon (GenBank: JF964960.1), annotations were generated in MITOchondrial genome annotation Server (MITOS) (Bernt et al. 2013).

The total length of O. andersonii was 18,297 bp (GenBank: MH844862). The base composition of the complete mitogenome was 34.20% A, 13.14% G, 25.37% T, and 27.23% C, with a slightly A + T bias of 59.57%. The complete mitochondrial genome included 13 protein-coding genes(PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, 2 control regions (CRs), and an origin of light-strand replication (OL), which is similar to the typical mtDNA of reported snakes (Xu et al. 2015; Sun et al. 2017). All protein-coding genes had 11,888 bp length for coding 3730 amino acid. Twelve protein-coding genes were encoded on heavy strand (H-strand) and only ND6 gene on the light strand. Most protein-coding genes initiated with ATT or ATG except for ND4L and ND6, which began with GTG or ATA. Eight protein-coding genes terminated with the complete stop codon TAA (ND1, ND2, ATP8, ATP6, ND4L, ND4, ND5 and Cytb), ND6 regarded AGG as a stop codon, and the rest showed an incomplete stop codon T––. The size of the 22 tRNA genes ranged from 57 (tRNA^Ser-AGY) to 73 bp (tRNA^Leu-UUR). The O_L was 35 bp in length, located between tRNA^Asn and tRNA^Cys genes. The 12s rRNA and 16s rRNA were 924 and 1461 bp, respectively, separated by the tRNA^Val.

To validate the phylogenetic relationship of O. andersonii within the Colubridae, the Maximum Likelihood tree was constructed by MEGA 7.0 (Kumar S et al. 2016) using 13 PCGs from O. andersonii, the other related 18 species were from the family Colubridae, and one outgroup from Elapidae (Figure 1). The ML tree showed that O. andersonii clustered with the Thermophis baileyi and Nerodia sipedon, highly supported by a bootstrap value of 99%, and then clustered with Hebius vibakari ruthveni and Rhabdophis tigrinus, which was in accordance with recent molecular works (Wang et al. 2017). The mitogenome obtained in this study enriches the genomic resources available for further evolutionary studies, and can provide genomic resources for Cloubridae studies.

Figure 1. Maximum Likelihood tree for 20 species based on mitogenome PCGs sequences. GenBank accession numbers indicate the respective species name. The numbers on each node branches are bootstrap values.

Disclosure statement

The authors report no conflicts of interest and are solely responsible for the content and the writing of the paper.

Additional information

Funding

This project was supported by the Planning Funds of Scientific and Technology of Guangdong Province [2016B070701016], Local Survey of Terrestrial Wild Flora and Fauna Resources in Guangzhou [SYZFCG-[2017]032] and GDAS Special Project of Science and Technology Development [2017GDASCX-0107 and 2018GDASCX-0107].