http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, 2014; 25(4): 247–248 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2013.796518
MITOGENOME ANNOUNCEMENT
Next generation sequencing yields the complete mitochondrial genome of the scarce blue-tailed damselfly, Ischnura pumilio M. Olalla Lorenzo-Carballa1,2, David J. Thompson1, Adolfo Cordero-Rivera2, and Phillip C. Watts1
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1
Department of Evolution, Ecology and Behaviour, Institute of Integrative Biology, University of Liverpool, Liverpool, UK, and 2Evolutionary Ecology and Conservation Group, Department Ecology and Animal Biology, Universidad de Vigo, EUI Forestal, Campus Universitario A Xunqueira s/n, Pontevedra, Spain Abstract
Keywords
We report the entire mitochondrial genome of the scarce blue-tailed damselfly, Ischnura pumilio (Odonata, Coenagrionidae), using next-generation sequencing on genomic DNA. A de novo assembly provided a single contiguous sequence of 15,250 bp that contained the A þ T-rich region and all standard coding regions; gene configuration is similar to other odonates and comprises 13 protein-coding genes, two rRNA genes (12 S and 16 S rRNA) and 22 tRNA genes. We found a unique intergenic spacer in I. pumilio and confirm that the intergenic spacer s5 likely represents a synapomorphy between Anisoptera and Zygoptera. This is the first mitogenome sequence obtained for a member of the Coenagrionidae and demonstrates how next-generation sequencing technology can obtain mtDNA genome sequences without prior sample processing or primer design.
Damselfly, intergenic spacer, mitochondrial genome, Odonata
Odonata (dragonflies and damselflies) is a diverse Order of insects that comprises more than 5500 species and which occupies a key phylogenetic position as a putative basal-winged insect (Pterygota) (Lin et al., 2010; Simon et al., 2009). Obtaining complete mitochondrial genomes in a range of insect taxa would aid more robust phylogenetic reconstructions, but the complete mitochondrial genomes of only a handful of odonates are available: Davidius lunatus (GenBank: EU591677, Lee et al., 2009), Euphaea formosa (GenBank: HM126547, Lin et al., 2010), Pseudolestes mirabilis (GenBank: FJ606784, unpublished) and an incomplete genome of Orthetrum triangulare melania (GenBank: AB126005, Yamauchi et al., 2004). We used next-generation sequencing to obtain the complete mitochondrial genome of the damselfly Ischnura pumilio (Odonata, Coenagrionidae). Coenagrionids represent perhaps the best-studied odonate family, with representative species used as models of behavior (Thompson et al., 2011), senescence (Sherratt et al., 2010), colour polymorphism (Cordero, 1990) and sperm competition (Cooper et al., 1996). Total genomic DNA was extracted from the thorax muscle of a single I. pumilio, collected in UK, using the Qiagen DNAeasy kit and submitted for sequencing on 1/12 plate of a GS454 Flx (Roche) at the Centre for Genomic Research (http://www.liv. ac.uk/cgr/). Contig assembly was performed using CLC Genomics Workbench (CLC Bio, Denmark) with bubble size ¼ 150, word size ¼ 25 and default values for the remaining parameters. The mitogenome was annotated in Geneious v.6.0.5 (http://www.geneious.com/) by comparing with the available odonate mitogenomes (listed above) and with gene identify and open-reading Correspondence: Phillip C. Watts, Department of Evolution, Ecology and Behaviour, Institute of Integrative Biology, University of Liverpool, BioSciences Building, Crown Street, Liverpool L69 7ZB, UK. Tel: 0151 795 4524. Fax: 0151 795 4408. E-mail:
[email protected]
History Received 10 April 2013 Accepted 13 April 2013 Published online 24 June 2013
frames confirmed by BLAST (Altschul et al., 1990) searches against GenBank nr database (http://www.ncbi.nih.gov/). Transfer RNA genes were identified using tRNAscan-SE Search Server v.1.21 using a tRNA covariance model (Lowe & Eddy, 1997). Here, we provide the first mitogenome of a member of the Coenagrionidae and confirm the wider utility of 454 sequencing to obtain insect mitogenomes. The complete mitochondrial DNA sequence of I. pumilio (GenBank submission ID: 1619669) is 15,250 bp long, comparable with the other three complete odonate mitochondrial genomes (15,700 bp in E. formosa, 15,122 bp in P. mirabilis, and 15,913 bp in D. lunatus). It comprises the expected metazoan panel of 13 protein-coding genes, 2 rRNA genes (12 S and 16 S rRNA) and 22 tRNA genes (Table 1). Twelve protein-coding genes in I. pumilio employ the standard invertebrate mitochondrial start codons: five (cox2, cox3, cob, nad4 and nad4L) use ATG, four (nad1, nad3, atp8 and atp6) use TTG, two (nad2 and nad5) use ATT, and nad6 uses ATC; cox1 initiates with the non-canonical start codon TTA. Nine of the protein genes have the standard stop codons TAA (cob, nad1, nad2, nad4, nad4L, nad5, nad6 and atp6) and TAG (nad3). The cox1, cox2, cox3 and atp8 genes have an incomplete stop codon of a single T (Table 1). Base frequency of the whole coding mtDNA region is A ¼ 40.1%, T ¼ 32.6%, C ¼ 14.7% and G ¼ 12.6%, with an overall A þ T content of 72.7% that is within the range of values observed in the other odonate genomes. All protein-coding genes are AT-biased (73.3%), with atp8 having the highest (82.5%) and cox1 (66.6%) the lowest values; cox1 also has the lowest AT content in other odonates, but the protein-coding genes with the highest AT content differ between species. The tRNA sequences in I. pumilio range in size from 64 bp in trnC, trnR and trnS-TGA to 72 bp in trnK. The sequenced mtDNA genome of I. pumilio is identical in gene number and gene arrangement to that of the other four odonate mitogenomes. Fifteen gene junctions in the mtDNA of I. pumilio have short overlaps, with the largest junction
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Mitochondrial DNA, 2014; 25(4): 247–248
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Table 1. Organization of the mitochondrial genome of I. pumilio.
trnI trnQ trnM nad2 trnW trnC trnY s1 cox1 trnL1 cox2 trnK trnD atp8 atp6 cox3 trnG nad3 trnA trnR trnN trnS1 trnE trnF s2 nad5 trnH nad4 nad4L trnT s3 trnP nad6 cob trnS2 s4 nad1 trnL2 l-rRNA trnV s-rRNA A þ T-rich region
Strand
Position
Length
AT (%)
Anti/Start codon
Stop
þ – þ þ þ – – n.a. þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ – n.a. – – – – þ n.a. – þ þ þ n.a. – – – – –
1–67 65–132 132–200 201–1196 1195–1263 1256–1319 1320–1386 1387–1403 1404–2959 2962–3027 3028–3714 3716–3787 3787–3854 3852–4005 4004–4687 4687–5473 5474–5540 5541–5894 5893–5959 5959–6022 6026–6091 6091–6161 6162–6226 6227–6294 6295–6312 6313–8022 8023–8087 8092–9429 9423–9716 9719–9785 9786–9800 9801–9869 9872–10,387 10,387–11,520 11,519–11,582 11,583–11,599 11,600–12,550 12,552–12,619 12,619–13,908 13,909–13,979 13,982–14,761 14,762–15,250
67 68 69 996 69 64 67 17 1556 66 687 72 68 156 684 787 67 354 67 64 66 71 65 68 18 1710 65 1335 294 67 15 69 516 1134 64 17 951 68 1290 71 780 489
71.6 76.4 71.0 77.4 73.9 73.5 65.6 76.5 66.6 59.1 68.6 66.7 82.4 82.0 71.4 67.5 73.1 73.2 77.6 65.7 69.7 66.2 87.7 79.4 94.4 73.8 75.3 72.9 76.8 67.1 33.4 78.3 77.6 70.9 72.7 82.4 73.8 75.0 76.0 67.6 74.8 86.3
GAT CAT ATT TCA GCA GTA
– – – TAA – – –
TTA TAA ATG CTT GTC TTG TTG ATG TCC TTG TGC TCG GTT GCT TTC GAA
T(aa) – T(aa) – – T(aa) TAA T(aa) – TAG – – – – – –
ATT GTG ATG ATG TGT
TAA – TAA TAA –
TGG ATC ATG TGA
– TAA TAA –
TTG TAG – TAC –
TAA – – – –
The incomplete stop codons are labeled in the table with parentheses. s1–s4 are intergenic spacers.
8 nucleotides (between trnW and trnC). We found a unique spacer s2 between trnF and nad5 in I. pumilio. The s5 spacer shared between O. triangulare melania and D. lunatus was not found in I. pumilio or P. mirabilis, supporting the idea that this could be a synapomorphy for Zygoptera and Anisoptera (Lin et al., 2010).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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