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.