2.4 | Sequence assembly and sequence analysis
After quality control, clean data from complete mtDNA were aligned
against a reference genome (GenBank, NC_001788.1) using
Bowtie2. Reads on the calibration
were reserved and SPAdes v3.13.0 (Parameters: K 127) was used for genome
assembly. The splicing result was compared with the closed reference
genome using BLASTN, and the assembly result was determined accordingly.
tRNAs were recognized and their secondary structures were predicted by
tRNAscan-SE v2.0 (Chan & Lowe, 2019). The most variable region was
identified by sequence alignment against the reference genome. We
derived a circular map of the mitogenome using
ORDRAW (Greiner et al., 2019), and
base composition skew was calculated using AT-skew = (A − T)/(A + T) and
GC-skew = (G − C)/(G + C) (Perna & Kocher, 1995).
The 418 bp section was manually edited and aligned against the reference
sequence using CLUSTALX v2.0 (Larkin
et al., 2007). DNAsp v6 (Rozas et al., 2017) was used to calculate the
nucleotide diversity, polymorphic sites, and haplotype diversity of the
most variable region in the HGD mtDNA D-loop region. Genetic
relationships among populations were determined using median-joining
networks (MJN) using Network v.10.1.0.0 software (Bandelt et al., 1999),
the MJN was constructed from 11 haplotypes found in this study and 60
reference sequences found previously (Ozkan Unal et al., 2020; Xia et
al., 2019), the 15 sequences including Hap4, Hap6, Hap7, and Hap10,
Hap12, Hap16, Hap18, Hap20, Hap22, Hap23, Hap24, Hap25, Hap26, Hap27,
Hap29 belonged to Clade Ⅰ. Reference mtDNA D-loop region sequences of
wild and domestic donkey were obtained from GenBank and used as
comparators
of HGDs to build phylogenetic trees and speculate their origin (Han et
al., 2014; Ivankovic et al., 2002; Kimura et al., 2011; Oakenfull et
al., 2000; Ozkan Unal et al., 2020). The
phylogenetic tree was drawn using the
Neighbor-joining (NJ) method. Bootstrap values were estimated using
1,000 repetitions (Felsenstein, 1985), based on the Kimura-2-parameter
genetic distances (Saitou & Nei, 1987), and reconstructed using MEGA
v7.0 (Kumar et al., 2016).