Complete mitochondrial genome of the Cumberland slider (Trachemys scripta troostii, Testudienes: Emydidae) in Korea

Abstract The complete mitochondrial (mt) genome of Trachemys scripta troostii was sequenced and was characterized, which comprised 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding control region. Phylogenetic analysis based on the full mt genome indicated that T. s. troostii was more closely related to T. scripta from Canada than to T. s. elegans from China or T. s. scripta fom China. This is the first complete mt genome from T. s. troostii, which provides data for further study of phylogeny in Emydidae.

The Cumberland slider (Trachemys scripta troostii) is native in Cumberland and Tennessee Rivers, from south-eastern Virginia and Kentucky to north-eastern Alabama in the United States (Cagle 1950). Trachemys scripta was traded by pet market worldwideincluding Austria, Poland, and Korea, but some were deliberately released to the field (Kitowski 2013;Kleewein 2014;Koo et al. 2020). We collected the turtle which is not native in Korea. With some morphological characters such as the shape of head, and the color pattern of carapace and plastron bone, we identified it as T. s. troostii. According to a recent study, about 1.2% of non-native turtles corresponded to T. s. troostii in Korea (Koo et al. 2020). Compared to other T. scripta subspecies, there is lack of genetic research about T. s. troostii in the world. There was a phylogenetic study about T. s. troostii by using three mitochondrial (mt) DNA fragments (12 s ribosomal RNA, Cytochrome b þ 29 bp DNA coding for transfer RNA-Threonine, NADH-ubiquinone oxidoreductase chain 4 L/ NADH-ubiquinone oxidoreductase chain 4) (Vamberger et al. 2020). However, its complete mt genome has not been reported yet. We first sequenced the complete mt genome of T. s. troostii, and this data can be used for further phylogenetic and evolutionary studies.
The T. s. troostii specimen was collected from Gwangju (35 10 0 28.1 00 N; 126 54 0 35.9 00 E), Korea. We extracted the total genomic DNA from the tail using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) according to the manufacturer's protocol and the extracted DNA sample was deposited at the Museum of Wildlife, located in Research Center of Ecomimeitcs, Chonnam National University, Korea (Specimen accession number: 2020-RCE-TST001; shcol2002@chonnam.ac. kr). We constructed the complete mt genome by primer walking method with the primer pairs (Supplementary Table  1). The complete mt genome was sequenced using Applied Biosystems 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). We used Needleman-Wunsch algorithm on NCBI to align the sequences, checked quality control from chromatogram data provided by Bionics (Seoul, Korea) which offers DNA sequencing services. Each read was aligned and annotated by comparing T. scripta mt genome (Accession No. FJ392294), T. s. elegans mt genome (Accession No. KM216748), and T. s. scripta mt genome (Accession No. KM216749) in GenBank.
The complete mt genome of T. s. troostii was 16,810 bp in length deposited in GenBank (Accession No. MW122292), and contained 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes (12 s and 16 s), and a putative non-coding control region (NCR). Twelve PCGs, 14 tRNAs, 2 rRNAs were predicted to be located in the heavy strand, whereas 1 protein-coding gene (NADH dehydrogenase subunit 6) and 8 tRNAs were encoded in the light strand. The nucleotide composition of the T. s. troostii from Korea mt genome (A ¼ 34.3%, C ¼ 25.9%, G ¼ 12.9%, and T ¼ 27.0%) was same with that of T. scripta from Canada (A ¼ 34.3%, C ¼ 25.9%, G ¼ 12.9%, and T ¼ 27.0%), and similar with that of T. s. elegans from China (A ¼ 34.2%, C ¼ 25.9%, G ¼ 12.9%, and T ¼ 27.0%), and T. s. scripta from China (A ¼ 34.2%, C ¼ 25.9%, G ¼ 12.9%, and T ¼ 27.0%). The sequence comparison with T. s. troostii from Korea and T. scripta from Canada indicated a 99.97% sequence identity. But the sequence identity was 99.68% between T. s. troostii from Korea and T. s. elegans from China, and was 99.41% between T. s. troostii from Korea and T. s. scripta from China.
In order to investigate the phylogenetic position of T. s. troostii from Korea, the full mt genome sequences of 12 turtle species were extracted from Genbank. Based on other studies (Krenz et al. 2005;Russell and Beckenbach 2008), we chose Pelomedusa subrufa as an outgroup which belongs to the suborder 'Pleurodira.' The sequences were aligned using MUSCLE algorithm (Edgar 2004), and the phylogenetic tree was constructed using Maximum Likelihood (ML) method with Tamura-Nei model as the nucleotide substitution type in MEGA X software (Tamura and Nei 1993;Kumar et al. 2018). It shows that every T. scripta subspecies is clustered in a monophyletic group. T. s. troostii from Korea is more closely related to T. scripta from Canada than to T. s. elegans from China or T. s. scripta from China ( Figure 1). These data provide important molecular information for further studies about evolutionary analysis and can be used as a useful genetic marker for identification and ecological studies on T. s. troostii.

Disclosure statement
No potential conflict of interest was reported by the authors.  Data availability statement GenBank accession number from the complete mitochondrial genome of Trachemys scripta troostii (MW122292) has been registered with the NCBI database (https://www.ncbi.nlm.nih.gov/).