3 | RESULTS
3.1 | Structure and organization of complete mtDNA
We deposited the complete mtDNA − a circular 16 670 bp molecule − in GenBank under accession no. MZ911746. The gene map of Huaibei grey donkey mtDNA is presented in Figure 2. There were 13 protein coding genes (PCGs) including 7 NADH dehydrogenase complex subunits (ND1-6 and ND4L ), three cytochrome oxidase subunits (COX1-3 ), cytochrome b (CYTB ) and two ATPase subunits (ATP6 and ATP8 ). The mitogenome also contains two rRNA genes (rRNAL , rRNAS ), 22tRNA genes and a control region (D-loop). With respect to their location, 14 tRNA genes, 12 PCGs and two rRNA genes were located on the positive strand and the remaining eight tRNA genes and one PCG on the negative strand (Table 1). On the whole, a bias in nucleotide composition was observed toward A (32.3%) and T (25.6%), with respect to C (28.9%) and G (13.2%). We observed a negative GC-skew (–0.3729) and a positive AT-skew (+0.1157), indicating that A and C were marginally more numerous than T and G (Table 2).
3.2 | Condon usage and protein coding genes
The total length of the 13 protein coding genes (PCGs) was 9 911 bp, and they contained 57.3% AT nucleotides, with a positive AT-skew (+0.0750) and a negative GC-skew (–0.4239). Relative synonymous codon usage values for the Huaibei grey donkey were shown in Table 3. Codons encoding Trp were infrequent, while those encoding Leu and Ser occur most frequently (Figure 3). The PCGs region consisted of a total of 5 559 codons. The initiation and termination signals, as well as gene lengths, are listed in Table 2. Eleven PCGs had the ATG start codon, with the exception for ND2and ND3 , which had ATA as the start codon. Eight PCGs (ND1 , COX1 , COX2 , ATPase8 , ATPase6 ,ND4L , ND5 , and ND6 ) had TAA as the stop codon;ND2 , COX3 , and ND3 had TAG as the stop codon; andND4 and CYTB had AGA as the stop codon. The most frequently used amino acids by the PCGs of the mitogenomes of Huaibei grey donkey were leucine and serine (12.4% and 12.4%); among the 64 available codons, the three most frequently used codons are CUA (2.12%) for Leu2, UCA (3.18%) for Ser1, and GGA (2.41%) for Gly.
3.3 | Transfer and ribosomal RNA genes
The total length of the 22 tRNAs in Huaibei grey donkey mtDNA was 1 516 bp with an A + T content of 61.7%, positive AT-skew (+0.1183), and negative GC-skew (–0.1854). Most tRNA genes had the common cloverleaf secondary structure, excluding tRNA-Ser (GCT), which lacked the dihydrouridine arm (Figure 4). The total size of the two rRNAs was 2 555 bp, the A + T content was 60.1%, the AT-skew was positive (+0.2164), and the GC-skew was negative (–0.1508). The rrnL was located between trnV and trnL1 , and the rrnS was located between trnV and trnF . The length of the rrnL andrrnS was 1 580 bp and 975 bp, respectively.