The Complete Mitochondrial Genome and Phylogenetic Analysis of Chinese Jianchang Horse (Equus caballus)

The Complete Mitochondrial Genome and Phylogenetic Analysis of Chinese Jianchang Horse (Equus caballus) Xia Xiao1, Shizhong Yang2, Daijun Lin2, Yi Wang2, Yuxiang Hua1, Yan Wang1 and Linjie Wang1* 1Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, PR China 2Liangshan Institute of Animal Husbandry and Veterinary Science, Xichang 615042, PR China


PCR amplification and sequencing
Two microliters DNA was amplified in 30 or 35 cycles with specific primer pairs (Table 1) using the long and accurate DNA polymerase (PrimeSTAR® Max DNA Polymerase, TaKaRa, China). The primers were designed based on the Equus caballus (Accession No. X79547). PCR cycling conditions were as follows: 95°C initial denaturation for 4 min, 35 cycles of 95°C denaturation for 40 s, 60°C annealing for 40 s, and 72°C extension for 90 s. A final extension was performed at 72°C for 7 min. The PCR products were separated by electrophoresis in 2.0% agarose gel, and purified using a Gel Extraction Kit (Sangon, Shanghai, China). The purified products were subcloned into the pMD-18T vector (Takara, Japan) and sequenced by Beijing AuGCT Biotechnology Company. SeqMan software (DNASTAR Inc., USA) were employed to assemble a continuous sequence. DOGMA (http://dogma.ccbb.utexas. edu/) was used for annotating Jianchang horse mitochondrial genome. tRNA genes were defined with tRNAscan-SE 1.2 (http://lowelab.ucsc. edu/tRNAscan-SE/).

Phylogenetic analysis
The alignment of the nucleotide sequences of 11 mtDNA control regions of horses was performed with ClustalW (http://www.ebi.ac.uk/ clustalw/) using default settings. The phylogenetic tree was constructed using two methods: Maximum likelihood (ML) and Maximum parsimony (MP). ML analysis was performed with MEGA5 (http:// www.megasoftware.net/), based on mtDNA control regions of horses [9]. MP analysis was conducted using PAUP 4.0 Beta 10 program (http:// paup.csit.fsu.edu/about.html), with indels treated as missing character states. For the MP analysis, we performed using a heuristic search with the tree bisection and reconnection (TBR), and branch swapping algorithm. ML analysis was based on General Time Reversible model (GTR). The reliability of the resulting MP and ML tree topologies was tested using bootstrap analyses through 1 000 replicates for MP and 100 for ML.

Characters of Jianchang horse's mitochondrial genome
The complete mtDNA sequence of the Jianchang horse is composed of 16,614 bp, with 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes and one D-loop region ( Table 2), and has been deposited in GenBank (KT998647). The nucleotide composition (32.2% A, 28.5% C, 13.4% G, 25.9% T) is biased towards A-T content (58.1%), which is consistent with other horse breeds mitochondrial genomes [5,10,11]. The length of mitochondrial genome of Jianchang horse were shorter than that from Swedish horse (16,660 bp), and longer than that from Naqu Tibetan horse (16592 bp) [12]. There are variable numbers of 8 bp repeat fragments (ACCTGTGC) in control region among the breeds.
All protein-coding genes were found to be H-strand encoded, whereas ND6 was L-strand encoded. The initiation codons for ND2 and ND3 started with ATA, while the other genes had ATG start codon. There are four types of termination codon. The ND1 and ND2 genes are terminated with AGA. The COX3, ND3 and ND4 genes end with an incomplete termination codon of T, Cyt b ends with AGA, and the rest have a termination codon of TAA. In addition, compared with the Swedish horse mitochondrial genome (X79547), there are 105 nucleotide substitutions in the 13 protein-coding genes of the Jianchang horse.   The different horse breeds were classified into three main clusters according to the phylogenetic clades ( Figure 3). Obviously, Jianchang horse was significantly clustered as the clade with Debao and Yunnan horse. In addition, the Cheju and Deqin horse was clustered as another clade with Przewalskii and Zhongdian horse. Mongolian and Sanhe horse belongs to a cluster. Wuzhumuqin horse was clustered together with Xinihe and Naqu horse. Based on the mtDNA control region, cytochrome b diversity and microsatellite markers, previous studies have shown that Chinese horses have multiple maternal origins and high genetic diversity [13][14][15]. The Chinese breeds could be divided into five major groups by genotyping these animals for 27 microsatellite loci [13]. Previous study had also shown that there were two haplotypes of Y chromosome discovered in the domestic horse breeds in China [16], which develop into an important tool for horse population genetics [17].

Conclusions
In summary, we have sequenced the complete mtDNA sequence of the Jianchang horse. It has a typical mitogenome structure, containing 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes and and a non-coding control region (D-loop region). Data presented in our study provide a structural basis for future studies on mitogenome function in Jianchang horse. The phylogenetic tree analysis, therefore, will also contribute to the understanding the genetic diversity and origin of the Jianchang horse population in the future.  The phylogenetic tree of 11 horses includes that from Jianchang, Mongolian, Cheju, Yunnan, Zhongdian horse, Naqu horse, Deqin horse, Sanhe horse, Wuzhumuqin horse, Xinihe horse and Przewalskii horse based on D-loop sequence.