2. Materials and methods
2.1 Sample collection and genomic DNA extraction
Individuals of N. andersoni were collected from Lufeng County, Yunnan province, China (24°57′45.774″; 102°10′15.7296″, H=1875.43m), in August 2018. These individuals were sacrificed and dissected for organ collection. The heart, liver, spleen, lung, kidney and muscle were kept in the cryopreservation tubes directly. All the samples were immediately put in liquid nitrogen for short storage, then transported to the laboratory in dry ice and stored at -80°C. DNA was extracted from the muscle using mitochondrial extraction kit (Solarbio) and stored at -80°C.
2.2 Mitogenome sequencing, assembly and annotation
The mitochondrial DNA was subjected to random PCR (rPCR) as previously described [14]. The purified rPCR products were used to construct the sequencing library and sequenced on HiSeq-PE150 instrument (TIANGEN, Beijing, China). The raw reads were trimmed and filtered using Trimmomatic (Version 0.39) [15]. The cleaned reads were aligned to NCBI non-redundant protein sequence database using BLASTx by DIAMOND [16]. Mitochondrial reads were picked and de novo assembled into a complete mitochondrial genome using Geneious software package (Version 2019.1.1) [17]. Protein coding genes (PCGs) were annotated using the NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) and BLASTx with the vertebrate mitochondrial genetic code. The tRNA genes were identified using the tRNAscan-SE Search Server under the default search mode, using the vertebrate mitochondrial genetic code source [18]. Composition skew analysis was calculated according to the formulas: AT skew = (A-T)/ (A + T) and GC skew = (G-C)/ (G + C) [19]. Relative synonymous codon usage (RSCU) values were calculated using CodonW 1.4.2 [20]. The circular mitochondrial genome map ofN. andersoni was drawn using OGDRAW 1.3.1 [21].
2.3 Phylogenetic analysis
Phylogenetic analysis was performed comparing N. andersoni and 12 other rat mitogenomes downloaded from GenBank (Table 1). The nucleotide sequences were aligned using ClustalX with default settings before concatenation by DAMBE (Version 7.2) [22,23]. Models of evolution were evaluated using corrected Aikake Information Criteria (AICc) in jModelTest 2.1.10 to determine the best nucleotide substitution model [24]. Maximum likelihood (ML) analysis of the 13 PCGs in 13 species of rat was also performed using MEGA X [25]. The support values of the ML tree were evaluated via a bootstrap test with 1,000 iterations.