http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–2 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2014.1003847
MITOGENOME ANNOUNCEMENT
The complete mitochondrial genome of the flesh fly, Muscina stabulans (Diptera: muscidae) Lingmei Lan1, Ying Liu2, Jie Yan1, Lin Lin1, and Lagabaiyila Zha1 Department of Forensic Sciences, School of Basic Medicine, Central South University, Changsha, Hunan, China and 2Department of Oral and Maxillofacial Surgery, Xiangya Stomatological Hospital, Central South University, Changsha, Hunan, China
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 07/01/15 For personal use only.
1
Abstract
Keywords
In this study, we sequence the complete mitochondrial genome of Muscina stabulans (Diptera: Muscidae), a forensically important entomology, for the first time. The 15,933 bp circular genome contains the 37 genes found in a typical metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding A + T-rich region. All the protein initiation codons are ATN, except for cox1 and ND1 that begin with TCG and TTG. The arrangement of the genes was the same as that found in the other insect. The overall base composition on heavy strand was as follows A: 40.00%, G: 8.80%, C: 13.34%, T: 37.86% and the A + T content 77.86%. The mitochondrial genome of Muscina stabulans presented will be valuable for resolving phylogenetic relationships within the family Muscidae and order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.
A + T-rich region, forensic entomology, identification, tRNA, species
Muscina stabulans (Falle´n, 1987), the false stable fly, is a cosmopolitan species, which is common in the Neotropics (Carvalho et al., 2005; Patitucci et al., 2010). The flesh fly, Muscina stabulans, which belongs to the Muscina genus, Muscidae family and Diptera order, is an important forensic entomology (Benecke, 2001). With the main duty of finding the post-mortem interval, it was sequenced for the first time. In this research, we report the complete mitochondrial genome sequence of Muscina stabulans (GenBank accession number: KM676394).
History Received 22 November 2014 Accepted 5 December 2014 Published online 28 January 2015
Muscina stabulans was obtained in June 2014 from our laboratory culture. The culture was established from locally (Central South University, Hunan province, China) caught flies. Genomic DNA was isolated from individual fly using the DNeasyÔ Tissue Kit (QIAGEN Inc. Ltd., Shanghai, China). The entire Muscina stabulans mitochondrial genome was amplified in eight fragments (Nelson et al., 2012). The PCR amplifications were performed in 50 mL reaction volumes. This volume consisted of 7.5 ml of sterilized distilled water, 10 ml of
Table 1. Gene profile and organization of Parasarcophaga portschinskyi mitochondrial genome. Gene/element tRNA-Ile tRNA-Glu tRNA-Met ND2 tRNA-Trp tRNA-Cys tRNA-Tyr CO I tRNA-Leu(UUR) CO II tRNA-Lys tRNA-Asp ATP8 ATP6 CO III tRNA-Gly
Strand
Location (bp)
Size (bp)
H L H H H L L H H H H H H H H H
1–66 67–136 151–219 220–1233 1232–1299 1301–1362 1366–1430 1429–2962 2963–3028 3033–3713 3720–3790 3790–3855 3856–4017 4011–4688 4688–5479 5482–5547
66 70 69 1014 68 62 65 1534 66 681 71 66 162 678 792 66
Start coden
Stop coden
Anticoden (Location in bp) GAT (31–33) TTG (103–105) CAT (188–190)
ATT (M)
TAA TCA (1262–1264) GCA (1331–1333) GTA (1397–1399)
TCG (S)
T*
ATG (M)
TAA
TAA (2992–2994) CTT (3750–3752) GTC (3820–3822) ATT (M) ATG (M) ATG (M)
TAA TAA TAA TCC (5512–5514) (continued )
Correspondence: Lagabaiyila Zha, Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, China. Tel: +86 731 88195495. Fax: +86 731 82355414. E-mail: [email protected]
2
L. Lan et al.
Mitochondrial DNA, Early Online: 1–2
Table 1. Continued.
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 07/01/15 For personal use only.
Gene/element 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 nc
Strand
Location (bp)
Size (bp)
Start coden
Stop coden
H H H H H H L L L L L H L H H H L L L L L nc
5548–5901 5903–5968 5968–6030 6032–6096 6096–6163 6166–6230 6390–6457 6458–8177 8193–8256 8257–9885 9589–9885 9888–9952 9953–10,018 10,021–10,545 10,545–11,681 11,680–11,746 11,766–12,713 12,715–12,779 12,780–14,141 14,138–14,209 14,209–14,994 14,995–15,933
354 66 63 65 68 65 68 1720 64 1629 297 65 66 525 1137 67 948 65 1362 72 786 939
ATT (M)
TAA
Anticoden (Location in bp) TGC (5932–5934) TCG (5997–5999) GTT (6062–6064) GCT (6122–6124) TTC (6196–6198) GAA (6421–6423)
ATA (M)
T*
ATA (M) ATG (M)
T* TAA
GTG (8224–8226) TGT (9918–9920) TGG (9985–9987) ATT (M) ATG (M)
TAA TAG
TTG (U)
TAG
TGA (11,709–11,711) TAG (12,748–12,750) TAC (14,174–14,176)
H, heavy; L, light; nc, non-coding. T*, TAA stop codon is completed by the addition of 30 A residues to the mRNA.
MightyAmp PCR Buffer, 1 ml (10 pmol) of each primer, 0.5 ml of DNA template and 1.0 ml (1.25 U) of TaKaRa MightyAmp Taq polymerase (Takara). PCRs were performed with following cycling conditions: 94 C for 2 min; 35 cycles of 94 C for 10 s, 50 C for 50 s; and a final extension step of 72 C for 1.5 min. PCR products were sequenced on both strands by the commercial service (BGIWuhan, Wuhan, China). The mitochondrial genome size, the order and orientation of the genes, was the same as that of the inferred ancestral arthropod genome (Clary & Wolstenholme, 1985). The complete genome of Muscina stabulans was circular, 15,933 bp in length with 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region as in other insects (Table 1). The genome possessed 939 bp A + T-rich or putative region located between tRNA-Ile gene and 12S ribosomal RNA. Among the 13 protein-coding genes, 11 were identified with ATN as start codon coding for M except COI and ND1, which is coding for S and U different from the standard invertebrate mitochondrial code but the same with several researches (Nelson, 2012; Weigl, 2010; Zhe et al., 2013). The genome possessing the standard complement of 22 tRNA genes was found in most animal mitochondrial genomes. The phylogenetic relationships within the muscidae have been confined largely to the morphology of male genitalia. However, muscidae are difficult to identify because of their highly similar morphological appearance. Based on some suggestion that mtDNA sequences could be successfully employed to distinguish some species of the muscidae (Dsouli, 2011; Li, 2010; Oliveira et al., 2007), this research contributes to the dipteran mitochondrial genomes currently available and provides the first complete mitochondrial genome for the flesh fly, Muscina stabulans. In addition, this mitochondrial genome plays a significant part in the identification and brings more positive impact on bioresearch and forensic science application. The mitochondrial genome sequence may also be used to help identifying favorable molecular markers suitable for species identifications.
Acknowledgements We express our sincere thanks to Professor Jifeng Cai (Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, China), who kindly provided facilities to work.
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 was supported by Science Foundation for The Youth Scholars of Central South University (NO.120925), the Natural Science Foundation for the Youth (NO. 81302621).
References Benecke M. (2001). A brief history of forensic entomology. Forensic Sci Int 120:2–14. Carvalho CJB de, Couri MS, Pont AC, Pamplona D, Lopes SM. (2005). A catalogue of the Muscidae (Diptera) of the Neotropical Region. Zootaxa 860:1–282. 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. Dsouli N, Delsuc F, Michaux J, Stordeur E, Couloux A, Veuille M, Duvallet G. (2011). Phylogenetic analyses of mitochondrial and nuclear data in haematophagous flies support the paraphyly of the genus Stomoxys (Diptera: Muscidae). Infect Genet Evol 11:663–70. Li X, Cai JF, Guo YD, Wu KL, Wang JF, Liu QL, Wang XH, et al. (2010). The availability of 16S rRNA for the identification of forensically important flies (Diptera: Muscidae) in China. Trop Biomed 27:155–66. 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. Oliveira MT, Azeredo-Espin AM, Lessinger AC. (2007). The mitochondrial DNA control region of Muscidae flies: Evolution and structural conservation in a dipteran context. J Mol Evol 64:519–27. Patitucci LD, Mulieri PR, Mariluis JC, Schnack JA. (2010). The population ecology of Muscina stabulans (Falle´n) (Diptera: Muscidae) along an urban-rural gradient of Buenos Aires Argentina. Neotrop Entomol 39:441–6. Weigl S, Testini G, Parisi A. (2010). The mitochondrial genome of the common cattle grub Hypoderma lineatum. Med Vet Entomol 24: 329–35. Zhao Z, Su TJ, Chesters D, Wang SD, Ho SY, Zhu CD, Chen XL, Zhang CT. (2013). The mitochondrial genome of Elodia flavipalpis Aldrich (Diptera: Tachinidae) and the evolutionary timescale of Tachinid flies. PLoS One 8:e61814.
MITOGENOME ANNOUNCEMENT
The complete mitochondrial genome of the flesh fly, Muscina stabulans (Diptera: muscidae) Lingmei Lan1, Ying Liu2, Jie Yan1, Lin Lin1, and Lagabaiyila Zha1 Department of Forensic Sciences, School of Basic Medicine, Central South University, Changsha, Hunan, China and 2Department of Oral and Maxillofacial Surgery, Xiangya Stomatological Hospital, Central South University, Changsha, Hunan, China
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 07/01/15 For personal use only.
1
Abstract
Keywords
In this study, we sequence the complete mitochondrial genome of Muscina stabulans (Diptera: Muscidae), a forensically important entomology, for the first time. The 15,933 bp circular genome contains the 37 genes found in a typical metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding A + T-rich region. All the protein initiation codons are ATN, except for cox1 and ND1 that begin with TCG and TTG. The arrangement of the genes was the same as that found in the other insect. The overall base composition on heavy strand was as follows A: 40.00%, G: 8.80%, C: 13.34%, T: 37.86% and the A + T content 77.86%. The mitochondrial genome of Muscina stabulans presented will be valuable for resolving phylogenetic relationships within the family Muscidae and order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.
A + T-rich region, forensic entomology, identification, tRNA, species
Muscina stabulans (Falle´n, 1987), the false stable fly, is a cosmopolitan species, which is common in the Neotropics (Carvalho et al., 2005; Patitucci et al., 2010). The flesh fly, Muscina stabulans, which belongs to the Muscina genus, Muscidae family and Diptera order, is an important forensic entomology (Benecke, 2001). With the main duty of finding the post-mortem interval, it was sequenced for the first time. In this research, we report the complete mitochondrial genome sequence of Muscina stabulans (GenBank accession number: KM676394).
History Received 22 November 2014 Accepted 5 December 2014 Published online 28 January 2015
Muscina stabulans was obtained in June 2014 from our laboratory culture. The culture was established from locally (Central South University, Hunan province, China) caught flies. Genomic DNA was isolated from individual fly using the DNeasyÔ Tissue Kit (QIAGEN Inc. Ltd., Shanghai, China). The entire Muscina stabulans mitochondrial genome was amplified in eight fragments (Nelson et al., 2012). The PCR amplifications were performed in 50 mL reaction volumes. This volume consisted of 7.5 ml of sterilized distilled water, 10 ml of
Table 1. Gene profile and organization of Parasarcophaga portschinskyi mitochondrial genome. Gene/element tRNA-Ile tRNA-Glu tRNA-Met ND2 tRNA-Trp tRNA-Cys tRNA-Tyr CO I tRNA-Leu(UUR) CO II tRNA-Lys tRNA-Asp ATP8 ATP6 CO III tRNA-Gly
Strand
Location (bp)
Size (bp)
H L H H H L L H H H H H H H H H
1–66 67–136 151–219 220–1233 1232–1299 1301–1362 1366–1430 1429–2962 2963–3028 3033–3713 3720–3790 3790–3855 3856–4017 4011–4688 4688–5479 5482–5547
66 70 69 1014 68 62 65 1534 66 681 71 66 162 678 792 66
Start coden
Stop coden
Anticoden (Location in bp) GAT (31–33) TTG (103–105) CAT (188–190)
ATT (M)
TAA TCA (1262–1264) GCA (1331–1333) GTA (1397–1399)
TCG (S)
T*
ATG (M)
TAA
TAA (2992–2994) CTT (3750–3752) GTC (3820–3822) ATT (M) ATG (M) ATG (M)
TAA TAA TAA TCC (5512–5514) (continued )
Correspondence: Lagabaiyila Zha, Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, China. Tel: +86 731 88195495. Fax: +86 731 82355414. E-mail: [email protected]
2
L. Lan et al.
Mitochondrial DNA, Early Online: 1–2
Table 1. Continued.
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 07/01/15 For personal use only.
Gene/element 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 nc
Strand
Location (bp)
Size (bp)
Start coden
Stop coden
H H H H H H L L L L L H L H H H L L L L L nc
5548–5901 5903–5968 5968–6030 6032–6096 6096–6163 6166–6230 6390–6457 6458–8177 8193–8256 8257–9885 9589–9885 9888–9952 9953–10,018 10,021–10,545 10,545–11,681 11,680–11,746 11,766–12,713 12,715–12,779 12,780–14,141 14,138–14,209 14,209–14,994 14,995–15,933
354 66 63 65 68 65 68 1720 64 1629 297 65 66 525 1137 67 948 65 1362 72 786 939
ATT (M)
TAA
Anticoden (Location in bp) TGC (5932–5934) TCG (5997–5999) GTT (6062–6064) GCT (6122–6124) TTC (6196–6198) GAA (6421–6423)
ATA (M)
T*
ATA (M) ATG (M)
T* TAA
GTG (8224–8226) TGT (9918–9920) TGG (9985–9987) ATT (M) ATG (M)
TAA TAG
TTG (U)
TAG
TGA (11,709–11,711) TAG (12,748–12,750) TAC (14,174–14,176)
H, heavy; L, light; nc, non-coding. T*, TAA stop codon is completed by the addition of 30 A residues to the mRNA.
MightyAmp PCR Buffer, 1 ml (10 pmol) of each primer, 0.5 ml of DNA template and 1.0 ml (1.25 U) of TaKaRa MightyAmp Taq polymerase (Takara). PCRs were performed with following cycling conditions: 94 C for 2 min; 35 cycles of 94 C for 10 s, 50 C for 50 s; and a final extension step of 72 C for 1.5 min. PCR products were sequenced on both strands by the commercial service (BGIWuhan, Wuhan, China). The mitochondrial genome size, the order and orientation of the genes, was the same as that of the inferred ancestral arthropod genome (Clary & Wolstenholme, 1985). The complete genome of Muscina stabulans was circular, 15,933 bp in length with 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region as in other insects (Table 1). The genome possessed 939 bp A + T-rich or putative region located between tRNA-Ile gene and 12S ribosomal RNA. Among the 13 protein-coding genes, 11 were identified with ATN as start codon coding for M except COI and ND1, which is coding for S and U different from the standard invertebrate mitochondrial code but the same with several researches (Nelson, 2012; Weigl, 2010; Zhe et al., 2013). The genome possessing the standard complement of 22 tRNA genes was found in most animal mitochondrial genomes. The phylogenetic relationships within the muscidae have been confined largely to the morphology of male genitalia. However, muscidae are difficult to identify because of their highly similar morphological appearance. Based on some suggestion that mtDNA sequences could be successfully employed to distinguish some species of the muscidae (Dsouli, 2011; Li, 2010; Oliveira et al., 2007), this research contributes to the dipteran mitochondrial genomes currently available and provides the first complete mitochondrial genome for the flesh fly, Muscina stabulans. In addition, this mitochondrial genome plays a significant part in the identification and brings more positive impact on bioresearch and forensic science application. The mitochondrial genome sequence may also be used to help identifying favorable molecular markers suitable for species identifications.
Acknowledgements We express our sincere thanks to Professor Jifeng Cai (Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, China), who kindly provided facilities to work.
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 was supported by Science Foundation for The Youth Scholars of Central South University (NO.120925), the Natural Science Foundation for the Youth (NO. 81302621).
References Benecke M. (2001). A brief history of forensic entomology. Forensic Sci Int 120:2–14. Carvalho CJB de, Couri MS, Pont AC, Pamplona D, Lopes SM. (2005). A catalogue of the Muscidae (Diptera) of the Neotropical Region. Zootaxa 860:1–282. 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. Dsouli N, Delsuc F, Michaux J, Stordeur E, Couloux A, Veuille M, Duvallet G. (2011). Phylogenetic analyses of mitochondrial and nuclear data in haematophagous flies support the paraphyly of the genus Stomoxys (Diptera: Muscidae). Infect Genet Evol 11:663–70. Li X, Cai JF, Guo YD, Wu KL, Wang JF, Liu QL, Wang XH, et al. (2010). The availability of 16S rRNA for the identification of forensically important flies (Diptera: Muscidae) in China. Trop Biomed 27:155–66. 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. Oliveira MT, Azeredo-Espin AM, Lessinger AC. (2007). The mitochondrial DNA control region of Muscidae flies: Evolution and structural conservation in a dipteran context. J Mol Evol 64:519–27. Patitucci LD, Mulieri PR, Mariluis JC, Schnack JA. (2010). The population ecology of Muscina stabulans (Falle´n) (Diptera: Muscidae) along an urban-rural gradient of Buenos Aires Argentina. Neotrop Entomol 39:441–6. Weigl S, Testini G, Parisi A. (2010). The mitochondrial genome of the common cattle grub Hypoderma lineatum. Med Vet Entomol 24: 329–35. Zhao Z, Su TJ, Chesters D, Wang SD, Ho SY, Zhu CD, Chen XL, Zhang CT. (2013). The mitochondrial genome of Elodia flavipalpis Aldrich (Diptera: Tachinidae) and the evolutionary timescale of Tachinid flies. PLoS One 8:e61814.
