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Abstract The Przewalskis gazelle， Procapra przewalskii， is one of the most endangered species in China， and is now found only in a single small area around the Lake Qinghai. In this study， the complete mitochondrial genome of P. przewalskii was determined and annotated. The circular genome is 16 547bp long， containing 13 proteincoding genes， 22 transfer RNA genes， 2 ribosomal RNA genes， and a predicted control region. The overall base composition was 34.0% A， 24.7% C， 28.3% T， and 13.0% G， with a total A+T content of 62.3%. Phylogenetic analysis of all 19 Bovidae species indicated that P. przewalskii showed a close relationship to Procapra gutturosa. Our results provide a great deal of useful information on further studies for conservation biology of Przewalskis gazelle.
Key words Procapra przewalskii; Przewalskis gazelle; Conservation genetics; Mitochondrial genome
The genus Procapra in Bovidae family of Mammalia comprises three Asian gazelles including Mongolian gazelle （Procapra gutturosa）， Tibetan gazelle （Procapra picticaudata）， and Przewalskis gazelle （Procapra przewalskii）， and were distributed in the Central Asian steppes of Mongolia （as well as some parts of Siberia and China）， Tibetan plateau， and several locations near Qinghai Lake in China. As the rarest species of Procapra， Przewalskis gazelle has been listed on the International Union for Conservation of Nature （IUCN） red list as endangered （http：//www.iucnredlist.org）. Competition with domestic livestock and fencing of the natural habitat are becoming the major threats that aggravate the decreasing population size. Mitochondrial genome could provide plenty of information for species identification， population structure and phylogenetic analysis[1-2]. In this study， we analyzed and reported the complete mitochondrial genome of Przewalskis gazelle （P. przewalskii）. Furthermore， the phylogenetic relationships within Bovidae and phylogenetic position of P. przewalskii were evaluated using mitochondrial genome. This mitochondrial genome provides useful genetic data for guiding conservation and management of Przewalskis gazelle.
Materials and Methods
The total DNA was isolated from the blood of P. przewalskii collected from Gangcha County （Qinghai， China） and then was sheared to construct a genomic library with insert fragment， followed by endrepaired， Atailed， tagged and subjected to NGS on Illumina HiSeq 2500 Sequencing System （Illumina， CA， USA）. A total of 1.542 G paired reads with average length of 150 bp was produced. Highquality clean reads were obtained by removing poorquality sequences and filtering data with CLC Genomics Workbench v10 （CLC Bio， Aarhus， Denmark）. This was used to assemble the mitochondrial genome using the program MITObim v1.8[3] with that of P. gutturosa as an initial reference sequence. Then， the mitochondrial genome of P. przewalskii was annotated using Geneious （Biomatters Ltd.， Auckland， New Zealand）. Results and Analysis
The complete mitochondrial genome of P. przewalskii （GenBank accession number： MG798933） is 16 547 bp in size. This circular mitochondrial genome contained the typical set of 37 mitochondrial genes including 13 PCGs， 22 tRNA genes and 2 ribosomal RNA （rRNA） genes （Fig. 1）. In addition， P. przewalskii mitochondrial genome included a typical control region. The overall base composition was 34.0% A， 24.7% C， 28.3% T， and 13.0% G， with a total A+T content of 62.3%. Eight tRNAs and ND6 were encoded on the light strand， while the other genes were located on the heavy strand. ATG was found in most of the PCGs and was considered as the most prevalent start codons. The typical stop codon TAA was identified in majority of the PCGs， and another canonical stop codon AGA was found in CYTB gene. All 22 tRNAs with the length that range from 60 to 75 bp were scattered through the whole genome. The 1 121bp control region located between trnP and trnF comprised of some conserved functional blocks that are involved in the regulation of replication and transcription.
Discussion
In order to infer the phylogenetic position of P. przewalskii， we selected 19 Bovidae species and one Cervidae species （Cervus elaphus） as the outgroup to reconstruct the Bovidae phylogeny. MEGA[4] was performed to construct the phylogenetic trees with maximum likelihood （ML） method using 13 mitochondrial PCGs. As shown in Fig. 2， Bovidae was divided into three clades. As expected， Hippotraginae and Alcelaphinae were grouped together then clustered with Cephalophinae， and Bovinae was a standalone clade. The remaining subfamilies formed a clade. P. przewalskii was clustered with P. gutturosa as the sister species. The newly reported P. przewalskii mitochondrial genome could provide more genetic data to further study on the processes of microevolution and subspeciation and new conservation strategies of critically endangered Przewalskis gazelle.
The values at the nodes correspond to the bootstrapped maximum likelihood percentages.
References
[1] BOORE JL. Animal mitochondrial genomes[J]. Nucleic Acids Res， 1999， 27（8）： 1767-1780.
[2] SUN Y， JIANG Q， YANG C， et al. Characterization of complete mitochondrial genome of Dezhou donkey （Equus asinus） and evolutionary analysis[J]. Curr Genet， 2016， 62（2）： 383-390.
[3] HAHN C， BACHMANN L， CHEVREUX B. Reconstructing mitochondrial genomes directly from genomic nextgeneration sequencing readsa baiting and iterative mapping approach[J]. Nucleic Acids Res， 2013， 41： e129.
[4] KUMAR S， STECHER G， TAMURA K. MEGA7： Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol， 2016， 33（7）： 1870-1874.
Key words Procapra przewalskii; Przewalskis gazelle; Conservation genetics; Mitochondrial genome
The genus Procapra in Bovidae family of Mammalia comprises three Asian gazelles including Mongolian gazelle （Procapra gutturosa）， Tibetan gazelle （Procapra picticaudata）， and Przewalskis gazelle （Procapra przewalskii）， and were distributed in the Central Asian steppes of Mongolia （as well as some parts of Siberia and China）， Tibetan plateau， and several locations near Qinghai Lake in China. As the rarest species of Procapra， Przewalskis gazelle has been listed on the International Union for Conservation of Nature （IUCN） red list as endangered （http：//www.iucnredlist.org）. Competition with domestic livestock and fencing of the natural habitat are becoming the major threats that aggravate the decreasing population size. Mitochondrial genome could provide plenty of information for species identification， population structure and phylogenetic analysis[1-2]. In this study， we analyzed and reported the complete mitochondrial genome of Przewalskis gazelle （P. przewalskii）. Furthermore， the phylogenetic relationships within Bovidae and phylogenetic position of P. przewalskii were evaluated using mitochondrial genome. This mitochondrial genome provides useful genetic data for guiding conservation and management of Przewalskis gazelle.
Materials and Methods
The total DNA was isolated from the blood of P. przewalskii collected from Gangcha County （Qinghai， China） and then was sheared to construct a genomic library with insert fragment， followed by endrepaired， Atailed， tagged and subjected to NGS on Illumina HiSeq 2500 Sequencing System （Illumina， CA， USA）. A total of 1.542 G paired reads with average length of 150 bp was produced. Highquality clean reads were obtained by removing poorquality sequences and filtering data with CLC Genomics Workbench v10 （CLC Bio， Aarhus， Denmark）. This was used to assemble the mitochondrial genome using the program MITObim v1.8[3] with that of P. gutturosa as an initial reference sequence. Then， the mitochondrial genome of P. przewalskii was annotated using Geneious （Biomatters Ltd.， Auckland， New Zealand）. Results and Analysis
The complete mitochondrial genome of P. przewalskii （GenBank accession number： MG798933） is 16 547 bp in size. This circular mitochondrial genome contained the typical set of 37 mitochondrial genes including 13 PCGs， 22 tRNA genes and 2 ribosomal RNA （rRNA） genes （Fig. 1）. In addition， P. przewalskii mitochondrial genome included a typical control region. The overall base composition was 34.0% A， 24.7% C， 28.3% T， and 13.0% G， with a total A+T content of 62.3%. Eight tRNAs and ND6 were encoded on the light strand， while the other genes were located on the heavy strand. ATG was found in most of the PCGs and was considered as the most prevalent start codons. The typical stop codon TAA was identified in majority of the PCGs， and another canonical stop codon AGA was found in CYTB gene. All 22 tRNAs with the length that range from 60 to 75 bp were scattered through the whole genome. The 1 121bp control region located between trnP and trnF comprised of some conserved functional blocks that are involved in the regulation of replication and transcription.
Discussion
In order to infer the phylogenetic position of P. przewalskii， we selected 19 Bovidae species and one Cervidae species （Cervus elaphus） as the outgroup to reconstruct the Bovidae phylogeny. MEGA[4] was performed to construct the phylogenetic trees with maximum likelihood （ML） method using 13 mitochondrial PCGs. As shown in Fig. 2， Bovidae was divided into three clades. As expected， Hippotraginae and Alcelaphinae were grouped together then clustered with Cephalophinae， and Bovinae was a standalone clade. The remaining subfamilies formed a clade. P. przewalskii was clustered with P. gutturosa as the sister species. The newly reported P. przewalskii mitochondrial genome could provide more genetic data to further study on the processes of microevolution and subspeciation and new conservation strategies of critically endangered Przewalskis gazelle.
The values at the nodes correspond to the bootstrapped maximum likelihood percentages.
References
[1] BOORE JL. Animal mitochondrial genomes[J]. Nucleic Acids Res， 1999， 27（8）： 1767-1780.
[2] SUN Y， JIANG Q， YANG C， et al. Characterization of complete mitochondrial genome of Dezhou donkey （Equus asinus） and evolutionary analysis[J]. Curr Genet， 2016， 62（2）： 383-390.
[3] HAHN C， BACHMANN L， CHEVREUX B. Reconstructing mitochondrial genomes directly from genomic nextgeneration sequencing readsa baiting and iterative mapping approach[J]. Nucleic Acids Res， 2013， 41： e129.
[4] KUMAR S， STECHER G， TAMURA K. MEGA7： Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol， 2016， 33（7）： 1870-1874.