ATP6 Gene Research Articles

Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene.

Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient’s cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.

The complete mitochondrial genome and phylogenetic analysis of sugarcane (Saccharum spp. hybrids) line 15a-53

The complete mitogenome of Saccharum spp. hybrid 15a-53 was determined in this study, which contains two distinct circular chromosomes, Chromosome 1 and 2. The length of Chromosome 1 is 300,848 bp with the GC content of 43.93%, while Chromosome 2 is 144,713 bp in length with the GC content of 43.57%. In Chromosome 1, 7.14% of the genome (21,468 nucleotides) is coding DNA and 92.86% (279,380 nucleotides) are intergenic region, while in Chromosome 2, 8.20% of genome (11,865 nucleotides) are coding DNA and 91.80% (132,848 nucleotides) are intergenic region. Chromosome 1 contains 20 protein-coding genes (three atp genes, three ccm genes, two cox genes, one mat gene, one mtt gene, six nad genes, and four rps genes), and 21 non-coding genes (15 tRNA and six rRNAs), while in Chromosome 2, there are 13 protein-coding genes (four nad genes, three rps genes, two atp genes, one ccm gene, one cob gene, one cox gene, and one rpl gene) and five tRNA genes. Maximum Likelihood phylogenetic analysis indicated that 15a-53 is close to S. spp. hybrid ROC22, S. spp. hybrid FN15 and S. officinarum Khon Kaen 3. The complete mitochondrial genome herein will provide useful sequence information for phylogenetic and evolutionary studies for Saccharum and Poaceae.

Complete mitochondrial DNA sequence of the East Asian minnow, Pungtungia herzi (Actinopterygii: Cypriniformes)

Some populations of the East Asian minnow Pungtungia herzi Herzenstein, 1892, which are naturally distributed in western Japan, have declined and are considered endangered. However, P. herzi has become a domestic invasive species in eastern Japan. Thus, knowledge of genetic features and phylogenetic relationships of P. herzi is important for conservation of this species and understanding its impact on ecosystems. We analyzed the complete mitochondrial genome using next generation sequencing of the East Asian minnow P. herzi from Yodo River, Osaka Prefecture, Japan. The mitochondrial genome of P. herzi consists of a circular molecule of 16,599 bp that includes 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region. The heavy (H)-strand was predicted to have 12 PCGs, 14 tRNA, and two rRNA genes, while the light (L)-strand was predicted to contain one PCGs and eight tRNA genes. The average AT content was 57.68%. The genes ATP8 and ATP6, ATP6 and COIII, ND4L and ND4, and ND5 and ND6 shared seven, one, seven, and four nucleotides, respectively. The initiation codons ATG and GTG were found in 12 and one genes, respectively. The termination codons TAA, TAG, incomplete TA–, and single T–– were observed in nine, one, one, and two genes, respectively. All the tRNA genes possessed a cloverleaf secondary structure. The phylogenetic relationships inferred using 13 PCGs (based on the maximum likelihood) were consistent with previous studies that predicted interrelationships of Cypriniformes.

The complete mitochondrial genome of the African leaf butterfly Kallimoides rumia (Insecta: Lepidoptera: Nymphalidae)

The African leaf butterfly Kallimoides rumia Doubleday, 1849 (Nymphalidae), lives in the understory of Afrotropical primary forests. Genome skimming with Illumina sequencing of K. rumia permitted assembly of a complete circular mitogenome of 15,234 bp consisting of 80.9% AT nucleotides, 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region. Kallimoides rumia COX1 features an atypical start codon (CGA). Genes ATP6, COX1, COX2, ND2, ND4 and ND5 feature incomplete stop codons, completed by adding 3’ A residues to the mRNA. Phylogenetic reconstruction places K. rumia as a distinct lineage, not closely related to Kallima, consistent with previous phylogenetic hypotheses.

The complete mitochondrial genome of the hydrozoan jellyfish Spirocodon saltatrix (Cnidaria; Hydrozoa; Anthoathecata) with phylogeny analysis

In the present study, we sequenced and analyzed the complete mitochondrial genome of the hydrozoan jellyfish Spirocodon saltatrix. The mitochondrial genome was a linear form (15,752 bp long, 70.4% AT), consisting of 13 protein coding genes (cox1, cox2, atp8, atp6, cox3, nad2, nad5, nad6, nad3, nad4L, nad1, nad4, and cytB), two tRNAs (tRNA-Met and tRNA-Trp), and two rRNAs (12S and 16S). Mitochondrial gene arrangement of the S. saltatrix was completely identical to already-known mitochondrial genomes of hydrozoans. Molecular phylogenetic analysis using 13 protein-coding genes showed that S. saltatrix was closely related to the hydrozoan Clava multicornis.

The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions.

Cytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6−, the putative mutant copy with a 300 bp truncation at the 5’-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6+) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6+ copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6+ is expressed only in MF samples, while apt6− mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6− could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility.

The complete mitochondrial genome of the Columbia lance nematode, Hoplolaimus columbus, a major agricultural pathogen in North America

BackgroundThe plant-parasitic nematode Hoplolaimus columbus is a pathogen that uses a wide range of hosts and causes substantial yield loss in agricultural fields in North America. This study describes, for the first time, the complete mitochondrial genome of H. columbus from South Carolina, USA.MethodsThe mitogenome of H. columbus was assembled from Illumina 300 bp pair-end reads. It was annotated and compared to other published mitogenomes of plant-parasitic nematodes in the superfamily Tylenchoidea. The phylogenetic relationships between H. columbus and other 6 genera of plant-parasitic nematodes were examined using protein-coding genes (PCGs).ResultsThe mitogenome of H. columbus is a circular AT-rich DNA molecule 25,228 bp in length. The annotation result comprises 12 PCGs, 2 ribosomal RNA genes, and 19 transfer RNA genes. No atp8 gene was found in the mitogenome of H. columbus but long non-coding regions were observed in agreement to that reported for other plant-parasitic nematodes. The mitogenomic phylogeny of plant-parasitic nematodes in the superfamily Tylenchoidea agreed with previous molecular phylogenies. Mitochondrial gene synteny in H. columbus was unique but similar to that reported for other closely related species.ConclusionsThe mitogenome of H. columbus is unique within the superfamily Tylenchoidea but exhibits similarities in both gene content and synteny to other closely related nematodes. Among others, this new resource will facilitate population genomic studies in lance nematodes from North America and beyond.

The mitochondrial genome of Phyllodiaptomus tunguidus Shen & Tai, 1964 (Copepoda, Calanoida) and its phylogeny

The mitochondrial genome of Phyllodiaptomus diaptomus was sequenced and assembled via Next-Generation Sequencing (NGS) and iterative assembly with a reference seed. The genome is 16446 bp long, A + T biased (69.4%), with 13 protein-coding genes, 22 tRNAs, and 2 rRNA. All protein-coding genes are initiated by a typical “ATN” codon. ND1, ND2, ATP6 genes are terminated with “TAG”, the other 10 genes are terminated with “TAA”. This is the first complete mitogenome published in the Diaptomidae. It provides molecular information useful to a better understanding of the phylogeny of calanoids.

Complete mitochondrial genome of Eupteryx(Stacla) minusula (Hemiptera: Cicadellidae: Typhlocybinae) from China

The complete mitochondrial genome of the leafhopper species Eupteryx (Stacla) minusula (Hemiptera: Cicadellidae: Typhlocybinae) are sequenced and annotated. The mitochondrial genome is 16945 bp, and nucleotide composition of the whole mitogenome is highly A + T biased (A: 43.6%; T: 35.2%, G: 11.3%, C: 9.9%). 11 PCGs have ATN as the start codon, except for atp8 and nad5 genes have TTG. The conventional termination codons (TAA or TAG) occur in 11 PCGs, while cox2 and nad5 uses incomplete codon (T) as termination codon. The complete mitogenome sequence of Eupteryx (Stacla) minusula is available in the GenBank with accession number: MN910279.

Identification of ATPase6 gene mutation from cimahi clinical isolates

Diabetes mellitus is a mitochondrial disease, caused by ATP deficiency. ATP was produced by the OXPHOS system in the mitochondria. Mitochondrial ATPase6 was one of important enzyme in oxidative phosphorylation process of ATP synthesis. The aim of this research was to provide information of ATP6 gene mutation that correlated to DMT2. A pair of primers was designed by in silico study. Blood samples were taken from DMT2 patients. Blood cells were lysed to obtain DNA template. Amplification of ATP6 gene was done by Polymerase Chain Reaction (PCR) technique. The amplicon was analyzed by 1% agarose gel electrophoresis. The gel showed 0,7 kb band of amplicon. The nucleotide sequencing showed that the amplicon was 681 base pairs. Analysis of phylogenetic showed that the sequence was 94,08% identical to homo sapiens ATP6 gene. Homology analysis between ATP6 gene from genbank and ATP6 gene fragments showed that there was a mutation 8860A>G. Amino acid analysis showed that 8860A>G change the amino acid T112A. Interestingly. It both happened in all samples, and it is a haplogroup. This is mean, it need further research to convince 8860A>G related to DMT2.

Complete mitochondrial genome sequence of Empoascanara dwalata (Hemiptera: Cicadellidae: Typhlocybinae)

In this study, the complete mitochondrial genome of the leafhopper species Empoascanara dwalata (Hemiptera: Cicadellidae: Typhlocybinae) was sequenced and annotated, and the sequence is available in the GenBank with accession number: MT350235. The circular mitogenome with 15,271 bp in length, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one AT-rich region. The nucleotide composition is as follows: A (41.7%), T (34.7%), G (10.6%), and C (13.0%). 11 PCGs have ATN as the start codon, except for atp8 and nad5 genes have TTG. The conventional termination codons (TAA or TAG) occur in 11 PCGs, while cox2 and nad5 use incomplete codon (T––) as termination codon. Phylogenetic analysis using 13 PCGs showed that E. dwalata was clustered with three other Typhlocybine species, which was consistent with the conventional classification.

P0029GLUCOSYLCERAMIDE SYNTHASE INHIBITION PRESERVES MITOCHONDRIAL FUNCTION AND REDUCES REACTIVE OXYGEN DAMAGE IN THE JCK MOUSE MODEL OF POLYCYSTIC KIDNEY DISEASE

Abstract Background and Aims Polycystic kidney disease (PKD) is characterized by the formation and progressive growth of renal cysts ultimately leading to end-stage renal disease (ESRD). Previous work has shown that treatment of PKD mouse models with a glucosylceramide synthase inhibitor (GCSi) inhibits glycosphingolipid metabolism and blocks disease progression. Clinical trials to determine the efficacy of GCSi treatment for ADPKD are currently ongoing. ADPKD patient kidneys, as well as kidneys from in vivo mouse models of PKD, display defects in mitochondrial morphology and function, leading to reactive oxygen species (ROS) generation. Functional defects include decreased fatty acid oxidation and increased glycolysis that can promote cellular proliferation. To determine if glucosylceramide synthase inhibition ameliorates mitochondrial dysfunction, we assessed the impact of GCSi treatment on mitochondrial formation and function in the jck mouse model of PKD. Method Twenty-six-day old WT or jck mice were treated with vehicle or 60 mg/kg GCSi (Genz667161) in feed for 38 days prior to tissue harvest. Kidney mRNA expression was measured using RT-PCR. Kidney protein levels were measured by western blot. Mitochondrial DNA content was measured using real-time PCR. Oxidized DNA was detected by immunohistochemistry using anti-8OHdG antibodies. Oxidized proteins were measured using the Oxyblot system (manufacturer). Results Reduced cystogenesis following GCSi treatment was associated with preserved mitochondrial number in jck mice as evidenced by increased levels of mitochondrial DNA, increased mitochondrial proteins (Tom20, VDAC, SirT3, and MT-Co1), and elevated mRNA expression (Nd3, Cox3, and Atp8). Stabilization of mitochondrial number was accompanied by activation of pathways that promote mitochondrial biogenesis, including increased PGC1α protein and Ppargc1a and Tfam1. Downregulation of several antioxidant genes (Gpx6, Gstk1, Prdx3, and Sod2) were observed in untreated jck samples. Consistent with this, levels of oxidized DNA and oxidized proteins were increased in untreated jck samples. Treatment with GCSi partially reversed the downregulation of antioxidant genes and decreased the levels of oxidized DNA and proteins to near WT levels in jck tissues. We then compared the jck model to tissues derived from ADPKD patients. Western blots demonstrate a reduction in electron transport chain (CxI-CxV) and mitochondrial outer-membrane markers in both untreated jck and ADPKD samples compared to controls. Similarly, the mitochondrial genes Nd1, Nd2, Cox2, and Atp6 were reduced in untreated jck kidneys and ADPKD samples relative to controls. As previously mentioned, we observed a generalized loss of antioxidant gene expression in untreated jck kidneys. Consistent with this, there were increased levels of both oxidized DNA and oxidized protein in untreated jck samples; this increase in DNA and protein oxidation was mirrored in ADPKD patient samples. Conclusion Mitochondrial number and function were reduced in both untreated jck mouse kidneys and human ADPKD samples. Increased oxidative stress, as evidenced by increased levels of oxidized DNA and proteins, is evident in both jck and ADPKD tissues. In vivo treatment with a GCSi effectively reversed the observed increase in oxidative stress and inhibited disease progression in the jck model of ADPKD. Our data demonstrate that reduced kidney cyst growth following GCSi treatment is associated with preserved mitochondrial formation and function.

The complete mitochondrial genome of sugarcane (Saccharum spp.) variety FN15

The complete mitogenome of Saccharum spp. hybrid FN15 was successfully sequenced. It contains two distinct circular chromosomes, Chromosome 1 and Chromosome 2. The former is 301,533 bp in length with the GC content of 43.90%, and 7.12% of genome (21,468 nucleotides) are coding DNA while 92.88% of genome (280,065 nucleotides) are intergenic region. The latter is 144,744 bp in length with the GC content of 43.57%, and 8.20% of genome (11,865 nucleotides) are coding DNA and 91.80% of genome (132,879 nucleotides) are intergenic region. Besides, Chromosome 1 contains 22 protein-coding genes (four atp genes, three ccm genes, three cox genes, one mat gene, one mtt gene, six nad genes and four rps genes), and 21 non-coding genes (15 tRNA and six rRNAs), whereas in Chromosome 2, there are 13 protein-coding genes (two atp genes, one ccm gene, one cob gene, one cox gene, one rpl gene, four nad genes and three rps genes) and five tRNA genes. Maximum Likelihood phylogenetic analysis demonstrated that FN15 is close with S. spp. hybrid ROC22, S. officinarum Khon Kaen 3 and S. bicolor species. This complete mitochondrial genome will provide essential DNA molecular data for further phylogenetic and evolutionary analysis for Saccharum.

Characterization of the complete mitochondrial genome of Drawida gisti (Metagynophora, Moniligastridae) and comparison with other Metagynophora species

Here, the complete mitochondrial genome (mitogenome) of Drawida gisti was sequenced and compared with the mitogenomes of other Metagynophora species. The circular mitogenome was 14,648 bp in length and contained two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and 22 transfer RNA genes (tRNAs). The types of constitutive genes and the direction of the coding strand that appeared in Drawida mitogenome were identical to those observed in other Metagynophora species, except for a missing lengthy non-coding region. The conservative relationships between Drawida species were supported by the overall analyses of 13 PCGs, two rRNAs, and 22 tRNAs. A comparison of the Metagynophora mitogenomes revealed that the ATP8 gene possessed the highest polymorphism among the 13 PCGs and two rRNAs. Phylogenetic analysis suggested that the Moniligastridae contained Drawida, which is a primitive Metagynophora group. Our study provides a step forward toward elucidating the evolutionary linkages within Drawida and even Metagynophora.

Mitochondrial genome sequence of Phytophthora sansomeanaand comparative analysis of Phytophthora mitochondrial genomes.

Phytophthora sansomeana infects soybean and causes root rot. It was recently separated from the species complex P. megasperma sensu lato. In this study, we sequenced and annotated its complete mitochondrial genome and compared it to that of nine other Phytophthora species. The genome was assembled into a circular molecule of 39,618 bp with a 22.03% G+C content. Forty-two protein coding genes, 25 tRNA genes and two rRNA genes were annotated in this genome. The protein coding genes include 14 genes in the respiratory complexes, four ATP synthase genes, 16 ribosomal proteins genes, a tatC translocase gene, six conserved ORFs and a unique orf402. The tRNA genes encode tRNAs for 19 amino acids. Comparison among mitochondrial genomes of 10 Phytophthora species revealed three inversions, each covering multiple genes. These genomes were conserved in gene content with few exceptions. A 3' truncated atp9 gene was found in P. nicotianae. All 10 Phytophthora species, as well as other oomycetes and stramenopiles, lacked tRNA genes for threonine in their mitochondria. Phylogenomic analysis using the mitochondrial genomes supported or enhanced previous findings of the phylogeny of Phytophthora spp.

Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa.

Characterization of a novel and valuable CMS system in Brassicarapa. Cytoplasmic male sterility (CMS) is extensively used to produce F1 hybrid seeds in a variety of crops. However, it has not been successfully used in Chinese cabbage (Brassicarapa L. ssp. pekinensis) because of degeneration or temperature sensitivity. Here, we characterize a novel CMS system, BVRC-CMS96, which originated in B.napus cybrid obtained from INRAE, France and transferred by us to B.rapa. Floral morphology and agronomic characteristics indicate that BVRC-CMS96 plants are 100% male sterile and show no degeneration in the BC7 generation, confirming its suitability for commercial use. We also sequenced the BVRC-CMS96 and maintainer line 18BCM mitochondrial genomes. Genomic analyses showed the presence of syntenic blocks and distinct structures between BVRC-CMS96 and 18BCM and the other known CMS systems. We found that BVRC-CMS96 has one orf222 from 'Nap'-type CMS and two copies of orf138 from 'Ogu'-type CMS. We analyzed expression of orf222, orf138, orf261b, and the mitochondrial energy genes (atp6, atp9, and cox1) in flower bud developmental stages S1-S5 and in four floral organs. orf138 and orf222 were both highly expressed in S4, S5-stage buds, calyx, and the stamen. RNA-seq identified differentially expressed mRNAs and lncRNAs (long non-coding RNAs) that were significantly enriched in pollen wall assembly, pollen development, and pollen coat. Our findings suggest that an energy supply disorder caused by orf222/orf138/orf261b may inhibit a series of nuclear pollen development-related genes. Our study shows that BVRC-CMS96 is a valuable CMS system, and our detailed molecular analysis will facilitate its application in Chinese cabbage breeding.

The complete mitochondrial genome sequence and phylogenetic analysis of sugarcane (Saccharum spp.) cultivar ROC22

The complete mitogenome of Saccharum spp. hybrid ROC22 (a modern sugarcane cultivar) was sequenced and analyzed in the present study. Our results revealed that, unlike most plants, the complete mitogenome of sugarcane contains two distinct circular chromosomes, Chromosome 1 and Chromosome 2. The length of Chromosome 1 is 300,837 bp with the GC content of 43.93%, and the length of Chromosome 2 is 144,755 bp with the GC content of 43.57%. Apart from 16 tRNAs and six rRNAs non-coding genes, there are 20 protein-coding genes in Chromosome 1, including six nad genes, four rps genes, three atp genes, three ccm genes, two cox genes, one mat gene and one mtt gene. Chromosome 2 contains 18 genes, including five tRNA genes and 13 protein-coding genes (four nad genes, three rps genes, two atp genes, one ccm gene, one cob gene, one cox gene and one rpl gene). Phylogenetic analysis indicated that Saccharum spp. hybrid ROC22 is close with Saccharum officinarum Khon Kaen 3 and Sorghum bicolor species.

Next-generation sequencing yields the complete mitochondrial genome of the Capsala pricei Hidalgo, 1959 (Platyhelminthes: Monogenea) from South China Sea

The complete mitochondrial genome of the Capsala pricei (Monogenea: Capsalidae) collected from the sailfish (Istiophorus platypterus) was sequenced by the next-generation sequencing method. The mitogenome is 13,851 bp in length, includes 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region, with the atp8 gene being absent. The overall content of A + T is 69.26%, which is significantly higher than the C + G content (30.74%). C shows the lowest frequency (13.42%) among four bases. Phylogeography analysis showed a close relationship between the genus Capsala and Neobenedenia with high bootstrap value supported.

The complete mitochondrial genome of the jellyfish Aurelia coerulea (Cnidaria and Scyphozoa) with phylogenetic analysis

In this study, we sequenced the complete mitochondrial genome of the jellyfish Aurelia coerulea (A. coerulea). The mitochondrial genome was a linear form (16,748 bp long, 65.7% AT), consisting of 13 protein coding genes (PCGs) (cox1, cox2, atp8, atp6, cox3, nad2, nad5, nad6, nad3, nad4L, nad1, nad4, and cytB), 2 rRNAs (12S and 16S), and 2 tRNAs (trnM and trnW). Mitochondrial gene arrangement of the A. coerulea was identical when compared to already-known mitochondrial genomes of Aurelia species. The maximum-likelihood (ML) phylogenetic tree using of 13 mitochondrial proteins showed that A. coerulea collected from Korea waters was closely related to A. limbata.

The complete mitochondrial genome of Parathailocyba orla (Hemiptera: Cicadellidae: Typhlocybinae)

The complete mitochondrial genome of the leafhopper Parathailocyba orla (Dworakowska, 1977) was sequenced and annotated in this study. The mitochondrial genome is 15,382 bp long, and nucleotide composition of the whole mitogenome is highly A + T biased (A: 45.8%, T: 33.3%, G: 8.6%, C: 12.3%). 12 PCGs have ATN as the start codon, except for atp8 gene uses TTG. All protein-coding genes used TAA as stop codon, except for nad5, cox2 and cox3, which use incomplete T–– as stop codon. Phylogenetic analysis using 13 PCGs showed that P. orla was clustered with three Typhlocybinae species, which was consistent with the conventional classification.