http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–3 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.958675
MITOGENOME ANNOUNCEMENT
The complete mitochondria genome of Chrysomya pinguis (Diptera: Calliphoridae) Jie Yan, Huidan Liao, Kai Xie, and Jifeng Cai
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, Hunan, P.R. China
Abstract
Keywords
Chrysomya pinguis (Ch. pinguis) belongs to blowfly species of great medical and forensic importance. In this study, we report the complete mitochondrial genome of the Ch. pinguis for species identification. The 38 genes presented in the 15,838 bp circular genome has been found in a typical Metazoan genome, of which there are 23 transfer RNA genes, 13 protein-coding genes and 2 ribosomal RNA genes. In addition, there is a non-coding A + T-rich region between one and another tRNA-Ile. The array of the genes is similar to that discovered in other insects. The overall base compositions of A, G, C and T are 38.72%, 9.86%, 14.07% and 37.35% respectively. This study contributes to the availability of dipteran mitochondrial genomes and provides the first complete mitochondrial genome of Ch. pinguis for entomologists to identify diagnostic regions for species identifications and phylogenetic analysis.
Chrysomya pinguis, mitochondria genome, species identification
Chrysomya pinguis (Ch. Pinguis; Walker, 1858) is one of the blowflies distributed in tropic and subtropic areas, such as Thailand (Kittikhun et al., 2014), Malaysia (Omar et al., 2003) and China (Wang et al., 2008). It is reported to be closely related to not only many bacteria diseases but also forensic pathology comprising estimation of postmortem interval and deduction of death spot (Chen & Hung, 2004; Saigusa et al., 2005). Nevertheless, till now we are limited to molecular biology information about it. Viewing it as a potential marker in forensic
History Received 31 July 2014 Revised 6 August 2014 Accepted 23 August 2014 Published online 18 September 2014
science, we report the complete mitochondrial genome of the Ch. pinguis for species identification and phylogenetic analysis (GenBank accession number: KM244730). The blowfly of Ch. pinguis was obtained in June 2014 in Changsha, China. The full mitochondrial genome was amplified in eight fragments and the double stranded sequence was determined by prime walking within each long polymerase chain reaction (PCR) product. All of the fragments were amplified by TaKaRa MightyAmp Taq (Takara Co., Dalian, China). PCR program
Table 1. Gene profile and organization of Ch. pinguis mitochondrial genome. Gene element tRNA-Ile tRNA-Glu tRNA-Met ND2 tRNA-Sec tRNA-Cys tRNA-Tyr CO I tRNA-Leu(UUR) CO II tRNA-Lys tRNA-Asp ATP8 ATP6
Strand
Location (bp)
Size (bp)
H L H H H L L H H H H H H H
1–66 71–139 148–216 217–1231 1232–1299 1292–1355 1366–1432 1431–2964 2965–3030 3036–3723 3724–3794 3794–3860 3861–4025 4019–4696
66 69 69 1015 68 64 67 1534 66 688 71 67 165 678
Start codon
Stop codon
Anticodon (Location in bp) GAT (31–33) TTG (116–118) CAT (185–187)
ATT (M)
T* TCA (1262–1264) GCA (1325–1327) GTA (1399–1401)
TCG (M)
T*
ATG (M)
T*
TAA (2994–2996) CTT (3754–3756) GTC (3825–3827) ATT (M) ATG (M)
TAA TAA (continued )
Correspondence: Jifeng Cai, Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, P.R. China. Tel/Fax: +86 731 82355414. E-mail: [email protected]
2
J. Yan et al.
Mitochondrial DNA, Early Online: 1–3
Table 1. Continued
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Gene element CO III tRNA-Gly ND3 tRNA-Ala tRNA-Arg tRNA-Asn tRNA-Ser(AGN) tRNA-Glu tRNA-Phe ND5 tRNA-His ND4 ND4L tRNA-Thr tRNA-Pro ND6 Cytb tRNA-Ser(UCN) ND1 tRNA-Leu(CUN) 16S rRNA tRNA-Val 12S rRNA misc_feature tRNA-Ile misc_feature
Strand
Location (bp)
Size (bp)
Start codon
Stop codon
H H H H H H H H L L L L L H L H H H L L L L L nc H nc
4701–5489 5496–5560 5561–5914 5917–5981 5981–6044 6050–6121 6121–6190 6191–6257 6276–6342 6343–8062 8078–8142 8143–9481 9475–9771 9774–9838 9839–9904 9907–10,431 10,431–11,565 11,566–11,633 11,650–12,588 12,599–12,663 12,664–13,994 13,995–14,066 14,067–14,850 14,851–14,939 14,940–15,005 15,006–15,838
789 65 354 65 64 66 70 67 67 1720 65 1339 297 65 66 525 1135 68 939 65 1331 72 784 89 66 845
ATG (M)
TAA
ATT (M)
TAA
Anticodon (Location in bp) TCC (5526–5528) TGC (5946–5948) TCG (6010–6012) GTT (6086–6088) GCT (6147–6149) TTC (6221–6223) GAA (6308–6310)
ATT (M)
T*
ATG (M) ATG (M)
T* T*
GTG (8110–8112) TGT (9804–9806) TGG (9872–9874) ATT (M) ATG (M)
TAA T*
ATA (M)
T*
TGA (11,596–11,598) TAG (12,632–12,634) TAC (14,031–14,033) GAT (14,970–14,972)
H, heavy; L, light; nc, non-coding. T*, TAA stop codon is completed by the addition of 30 A residues to the mRNA.
was consisted of 94 C for 2 min, 35 cycles of 98 C for 10 s, 50 C for 30 s, 68 C for 2.5 min with a final extension at 68 C for 10 min. DNA fragments were sequenced on both strands by the commercial service (Transduction Bio Co. Ltd., Wuhan, China). The total 15,838 bp genome of Ch. pinguis contained genes usually present in arthropod (Table 1). The mitochondrial genome size, the order and orientation of the genes, are of no difference compared with that of the inferred ancestral arthropod genome (Clary & Wolstenholme, 1985). The genome possessed the standard complement of 23 tRNA genes including 2 tRNA-Ile and there are totally 845 bp A + T-rich or putative region located between the two tRNA-Ile genes. Twelve of the 13 protein-coding genes were identified with ATN as start codon coding for M except COI, which is the same to the former result (Zhong et al., 2014) but different from the standard invertebrate mitochondrial code (Nelson et al., 2012; Weigl et al., 2010). According to forensic cases, blowflies are basically the first ones showing in the cadaver and depositing eggs during few hours after death. Estimation of postmortem interval and deduction of death spot can be carried out through analyzing succession laws and growing process of the flies related to the dead body. However, there are difficulties in traditional morphological identification caused by rather similar characteristics of fly larvae between blowflies and by closely related species as developmental rates (VanLaerhoven, 2008) and diapause responses (Ash & Greenberg, 1975). Species identification techniques of molecular biology including molecular taxonomy (Smith & Baker, 2008) serve as a strong supplement for morphological identification. Mitochondrial DNA fragments test is popular in forensic entomology for postmortem interval estimation purpose. This research provides the entire genome of mitochondrial DNA of Ch. pinguis which is probably valuable to the database improvement of diptera and molecular marker selection for forensic species identification.
Acknowledgements The authors thank Yadong Guo and Lazhagabaiyila Zha (postdoctors of Central South University) for providing the Ch. pinguis specimen and efficient technical instruction.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study is supported by science foundation for the youth scholars of central south university (NO. 120973) and the fundamental research funds for the central universities of central south university.
References Ash N, Greenberg B. (1975). Developmental temperature responses of the sibling species Phaenicia sericata and Phaenicia pallescens. Ann Entomo Soc Am 68:197–200. Chen WY, Hung TH. (2004). Shiao molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan. J Med Entomol 41:47–57. Clary DO, Wolstenholme DR. (1985). The mitochondrial DNA molecular of Drosophila yakuba: Nucleotide sequence, gene organization, and genetic code. J Mol Evol 22:252–71. Kittikhun M, Tunwadee KK, Kom S. (2014). Species composition of carrion blow flies in northern Thailand: Altitude appraisal. Rev Inst Med Trop Sao Paulo 56:179–82. Nelson LA, Cameron SL, Yeates DK. (2012). The complete mitochondrial genome of the flesh fly, Sarcophaga impatiens Walker (Diptera: Sarcophagidae). Mitochondrial DNA 23:42–3. Omar B, Marwi MA, Sun CM. (2003). Distribution of medically important flies at various altitudes of Titiwangsa range near Kuala Lumpur, Malaysia. Trop Biomed 20:137–44. Saigusa K, Takamiya M, Aoki Y. (2005). Species identification of the forensically important flies in Iwate prefecture, Japan based on mitochondrial cytochrome oxidase gene subunit I (COI) sequences. Legal Med 7:175–8.
DOI: 10.3109/19401736.2014.958675
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Smith JA, Baker NC. (2008). Molecular genetic identification of forensically important flies in the UK. Forensic Sci Int Genet 1:620–2. VanLaerhoven SL. (2008). Blind validation of postmortem interval estimates using developmental rates of blow flies. Forensic Sci Int 180: 76–80. Wang JF, Li ZG, Chen YC, Chen QS, Yin XH. (2008). The succession and development of insects on pig carcasses and their significances in estimating PMI in south China. Forensic Sci Int 179:11–18.
Mitogenome of C. pinguis
3
Weigl S, Testini G, Parisi A. (2010). The mitochondrial genome of the common cattle grub, Hypoderma lineatum. Med Vet Entomol 24: 329–35. Zhong M, Wang X, Liu Q. (2014a). The complete mitochondrial genome of the scuttle fly, Megaselia scalaris (Diptera: Phoridae). Mitochondrial DNA. [Epub ahead of print]. doi:10.3109/19401736.2013.879651. Zhong M, Wang X, Liu Q. (2014b). The complete mitochondrial genome of the flesh fly, Boettcherisca peregrine (Diptera: Sarcophagidae). Mitochondrial DNA. [Epub ahead of print]. doi:10.3109/19401736. 2013.873925.
MITOGENOME ANNOUNCEMENT
The complete mitochondria genome of Chrysomya pinguis (Diptera: Calliphoridae) Jie Yan, Huidan Liao, Kai Xie, and Jifeng Cai
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, Hunan, P.R. China
Abstract
Keywords
Chrysomya pinguis (Ch. pinguis) belongs to blowfly species of great medical and forensic importance. In this study, we report the complete mitochondrial genome of the Ch. pinguis for species identification. The 38 genes presented in the 15,838 bp circular genome has been found in a typical Metazoan genome, of which there are 23 transfer RNA genes, 13 protein-coding genes and 2 ribosomal RNA genes. In addition, there is a non-coding A + T-rich region between one and another tRNA-Ile. The array of the genes is similar to that discovered in other insects. The overall base compositions of A, G, C and T are 38.72%, 9.86%, 14.07% and 37.35% respectively. This study contributes to the availability of dipteran mitochondrial genomes and provides the first complete mitochondrial genome of Ch. pinguis for entomologists to identify diagnostic regions for species identifications and phylogenetic analysis.
Chrysomya pinguis, mitochondria genome, species identification
Chrysomya pinguis (Ch. Pinguis; Walker, 1858) is one of the blowflies distributed in tropic and subtropic areas, such as Thailand (Kittikhun et al., 2014), Malaysia (Omar et al., 2003) and China (Wang et al., 2008). It is reported to be closely related to not only many bacteria diseases but also forensic pathology comprising estimation of postmortem interval and deduction of death spot (Chen & Hung, 2004; Saigusa et al., 2005). Nevertheless, till now we are limited to molecular biology information about it. Viewing it as a potential marker in forensic
History Received 31 July 2014 Revised 6 August 2014 Accepted 23 August 2014 Published online 18 September 2014
science, we report the complete mitochondrial genome of the Ch. pinguis for species identification and phylogenetic analysis (GenBank accession number: KM244730). The blowfly of Ch. pinguis was obtained in June 2014 in Changsha, China. The full mitochondrial genome was amplified in eight fragments and the double stranded sequence was determined by prime walking within each long polymerase chain reaction (PCR) product. All of the fragments were amplified by TaKaRa MightyAmp Taq (Takara Co., Dalian, China). PCR program
Table 1. Gene profile and organization of Ch. pinguis mitochondrial genome. Gene element tRNA-Ile tRNA-Glu tRNA-Met ND2 tRNA-Sec tRNA-Cys tRNA-Tyr CO I tRNA-Leu(UUR) CO II tRNA-Lys tRNA-Asp ATP8 ATP6
Strand
Location (bp)
Size (bp)
H L H H H L L H H H H H H H
1–66 71–139 148–216 217–1231 1232–1299 1292–1355 1366–1432 1431–2964 2965–3030 3036–3723 3724–3794 3794–3860 3861–4025 4019–4696
66 69 69 1015 68 64 67 1534 66 688 71 67 165 678
Start codon
Stop codon
Anticodon (Location in bp) GAT (31–33) TTG (116–118) CAT (185–187)
ATT (M)
T* TCA (1262–1264) GCA (1325–1327) GTA (1399–1401)
TCG (M)
T*
ATG (M)
T*
TAA (2994–2996) CTT (3754–3756) GTC (3825–3827) ATT (M) ATG (M)
TAA TAA (continued )
Correspondence: Jifeng Cai, Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, P.R. China. Tel/Fax: +86 731 82355414. E-mail: [email protected]
2
J. Yan et al.
Mitochondrial DNA, Early Online: 1–3
Table 1. Continued
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Gene element CO III tRNA-Gly ND3 tRNA-Ala tRNA-Arg tRNA-Asn tRNA-Ser(AGN) tRNA-Glu tRNA-Phe ND5 tRNA-His ND4 ND4L tRNA-Thr tRNA-Pro ND6 Cytb tRNA-Ser(UCN) ND1 tRNA-Leu(CUN) 16S rRNA tRNA-Val 12S rRNA misc_feature tRNA-Ile misc_feature
Strand
Location (bp)
Size (bp)
Start codon
Stop codon
H H H H H H H H L L L L L H L H H H L L L L L nc H nc
4701–5489 5496–5560 5561–5914 5917–5981 5981–6044 6050–6121 6121–6190 6191–6257 6276–6342 6343–8062 8078–8142 8143–9481 9475–9771 9774–9838 9839–9904 9907–10,431 10,431–11,565 11,566–11,633 11,650–12,588 12,599–12,663 12,664–13,994 13,995–14,066 14,067–14,850 14,851–14,939 14,940–15,005 15,006–15,838
789 65 354 65 64 66 70 67 67 1720 65 1339 297 65 66 525 1135 68 939 65 1331 72 784 89 66 845
ATG (M)
TAA
ATT (M)
TAA
Anticodon (Location in bp) TCC (5526–5528) TGC (5946–5948) TCG (6010–6012) GTT (6086–6088) GCT (6147–6149) TTC (6221–6223) GAA (6308–6310)
ATT (M)
T*
ATG (M) ATG (M)
T* T*
GTG (8110–8112) TGT (9804–9806) TGG (9872–9874) ATT (M) ATG (M)
TAA T*
ATA (M)
T*
TGA (11,596–11,598) TAG (12,632–12,634) TAC (14,031–14,033) GAT (14,970–14,972)
H, heavy; L, light; nc, non-coding. T*, TAA stop codon is completed by the addition of 30 A residues to the mRNA.
was consisted of 94 C for 2 min, 35 cycles of 98 C for 10 s, 50 C for 30 s, 68 C for 2.5 min with a final extension at 68 C for 10 min. DNA fragments were sequenced on both strands by the commercial service (Transduction Bio Co. Ltd., Wuhan, China). The total 15,838 bp genome of Ch. pinguis contained genes usually present in arthropod (Table 1). The mitochondrial genome size, the order and orientation of the genes, are of no difference compared with that of the inferred ancestral arthropod genome (Clary & Wolstenholme, 1985). The genome possessed the standard complement of 23 tRNA genes including 2 tRNA-Ile and there are totally 845 bp A + T-rich or putative region located between the two tRNA-Ile genes. Twelve of the 13 protein-coding genes were identified with ATN as start codon coding for M except COI, which is the same to the former result (Zhong et al., 2014) but different from the standard invertebrate mitochondrial code (Nelson et al., 2012; Weigl et al., 2010). According to forensic cases, blowflies are basically the first ones showing in the cadaver and depositing eggs during few hours after death. Estimation of postmortem interval and deduction of death spot can be carried out through analyzing succession laws and growing process of the flies related to the dead body. However, there are difficulties in traditional morphological identification caused by rather similar characteristics of fly larvae between blowflies and by closely related species as developmental rates (VanLaerhoven, 2008) and diapause responses (Ash & Greenberg, 1975). Species identification techniques of molecular biology including molecular taxonomy (Smith & Baker, 2008) serve as a strong supplement for morphological identification. Mitochondrial DNA fragments test is popular in forensic entomology for postmortem interval estimation purpose. This research provides the entire genome of mitochondrial DNA of Ch. pinguis which is probably valuable to the database improvement of diptera and molecular marker selection for forensic species identification.
Acknowledgements The authors thank Yadong Guo and Lazhagabaiyila Zha (postdoctors of Central South University) for providing the Ch. pinguis specimen and efficient technical instruction.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study is supported by science foundation for the youth scholars of central south university (NO. 120973) and the fundamental research funds for the central universities of central south university.
References Ash N, Greenberg B. (1975). Developmental temperature responses of the sibling species Phaenicia sericata and Phaenicia pallescens. Ann Entomo Soc Am 68:197–200. Chen WY, Hung TH. (2004). Shiao molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan. J Med Entomol 41:47–57. Clary DO, Wolstenholme DR. (1985). The mitochondrial DNA molecular of Drosophila yakuba: Nucleotide sequence, gene organization, and genetic code. J Mol Evol 22:252–71. Kittikhun M, Tunwadee KK, Kom S. (2014). Species composition of carrion blow flies in northern Thailand: Altitude appraisal. Rev Inst Med Trop Sao Paulo 56:179–82. Nelson LA, Cameron SL, Yeates DK. (2012). The complete mitochondrial genome of the flesh fly, Sarcophaga impatiens Walker (Diptera: Sarcophagidae). Mitochondrial DNA 23:42–3. Omar B, Marwi MA, Sun CM. (2003). Distribution of medically important flies at various altitudes of Titiwangsa range near Kuala Lumpur, Malaysia. Trop Biomed 20:137–44. Saigusa K, Takamiya M, Aoki Y. (2005). Species identification of the forensically important flies in Iwate prefecture, Japan based on mitochondrial cytochrome oxidase gene subunit I (COI) sequences. Legal Med 7:175–8.
DOI: 10.3109/19401736.2014.958675
Mitochondrial DNA Downloaded from informahealthcare.com by Kainan University on 04/17/15 For personal use only.
Smith JA, Baker NC. (2008). Molecular genetic identification of forensically important flies in the UK. Forensic Sci Int Genet 1:620–2. VanLaerhoven SL. (2008). Blind validation of postmortem interval estimates using developmental rates of blow flies. Forensic Sci Int 180: 76–80. Wang JF, Li ZG, Chen YC, Chen QS, Yin XH. (2008). The succession and development of insects on pig carcasses and their significances in estimating PMI in south China. Forensic Sci Int 179:11–18.
Mitogenome of C. pinguis
3
Weigl S, Testini G, Parisi A. (2010). The mitochondrial genome of the common cattle grub, Hypoderma lineatum. Med Vet Entomol 24: 329–35. Zhong M, Wang X, Liu Q. (2014a). The complete mitochondrial genome of the scuttle fly, Megaselia scalaris (Diptera: Phoridae). Mitochondrial DNA. [Epub ahead of print]. doi:10.3109/19401736.2013.879651. Zhong M, Wang X, Liu Q. (2014b). The complete mitochondrial genome of the flesh fly, Boettcherisca peregrine (Diptera: Sarcophagidae). Mitochondrial DNA. [Epub ahead of print]. doi:10.3109/19401736. 2013.873925.
