Mitochondrial DNA, 2013; 24(2): 9496

MITOGENOME ANNOUNCEMENT

The complete mitochondrial DNA sequence of Crotalus horridus (timber rattlesnake)

JACOB B. HALL*, VINCENT A. COBB, & A. BRUCE CAHOON

Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA
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(Received 2 August 2012; accepted 17 August 2012)

Abstract The complete mitogenome of the timber rattlesnake (Crotalus horridus) was completed using Sanger sequencing. It is 17,260 bp with 13 protein-coding genes, 21 tRNAs, two rRNAs and two control regions. Gene synteny is consistent with other snakes with the exception of a missing redundant tRNA Ser. This mitogenome should prove to be a useful addition of a well-known member of the Viperidae snake family.

Keywords: Crotalus horridus, Viperidae, timber rattlesnake, mitogenome

Report The mitogenome of the timber rattlesnake (Crotalus horridus; order: Squamata, suborder: Serpentes, family: Viperidae) has been sequenced using oral tissue taken from a specimen collected in Rutherford County, Tennessee, USA (358 510 N, 868 180 W) on 20 October 2007. This specimen (APSU 19297) is deposited in the Museum of Zoology, Austin Peay State University, Clarksville, TN, USA. The sequence is deposited in GenBank as accession number HM641837. Primers were initially designed based on members of the snake subfamily Crotalinae: Agkistrodon piscivorus (DQ523161), Deinagkistrodon acutus (DQ343647), Gloydis blomhof (EU913477) and Ovophis okinavensis (AB175670). Additional primers were made using the newly collected C. horridus sequences. The primer set used to complete this mitogenome should be a useful starting point for the sequencing of other members of the family Viperidae, and is available upon request. Overall, 247 successful

sequencing runs were collected for a total of . 123,500 bases for 7X average depth of coverage. The Dual Organellar Genome Annotator (DOGMA; Wyman et al. 2004) was used to begin the annotation process. Start and stop codons of all proteincoding genes were located and/or conrmed using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) and Virtual Ribosome (Wernersson 2006). tRNAs were initially identied using DOGMA and tRNAscan-SE (Lowe and Eddy 1997) and checked individually using Sequenchers alignment features or by predicting secondary structure using mFold (Zuker 2003). Other mitochondria-specic features such as control regions and origin of light-strand replication were found using existing snake mitochondrial genomes and Sequenchers alignment features. The C. horridus mitochondrial genome (GenBank accession no. HM641837) is 17,260 bp with 13 protein-coding genes, 21 tRNAs, two rRNAs and two control regions (Figure 1). One exceptional feature is a missing tRNA Ser gene typically between tRNA His and tRNA Leu. This region was screened

Correspondence: A. Bruce Cahoon, Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA. Tel: 615 494 8792. Fax: 615 898 5093. E-mail: acahoon@mtsu.edu *Current address: Center for Human Genetics Research, 519 Light Hall, Vanderbilt University, Nashville, TN 37232, USA.
ISSN 1940-1736 print/ISSN 1940-1744 online q 2012 Informa UK, Ltd. DOI: 10.3109/19401736.2012.722999

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Figure 1. Gene content and order of the C. horridus mitochondrial genome. C. horridus is represented as a linear array of labeled boxes (genes) that are not drawn to scale. Boxes above the central line represent genes expressed from the heavy-strand promoters (CR1 and CR2, left-facing arrows) while boxes below the line represent those expressed from the light strand. The stem-loop structure (right-facing arrow) represents a secondary structure important for light-strand replication. The mtDNA of C. horridus closest taxonomic relative with a completed genome, A. piscivorus (Jiang et al. 2007) as well as the composite typical snake and typical vertebrate genomes (Dong and Kumazawa 2005) are shown for comparison. Snake mitogenomes differ from the typical vertebrate by the addition of a control region and the absence of a tRNA Leu. C. horridus differs from the typical snake mitogenome due to the absence of a tRNA Ser found in the HSL cluster between the nad4 and nad5 protein-coding genes. Both C. horridus and A. piscivorus have an additional tRNAPro not found in the typical vertebrate or typical snake synteny.

using DOGMA and tRNAscan-SE, neither of which recognized a tRNA. Also, secondary structure, as predicted by mFold, suggested that no potential tRNA structures were possible from the sequence. The gene synteny of vertebrate mitochondrial genomes, as a group, is relatively conserved, especially when compared with other metazoans (Gissi et al. 2008). In spite of this, it is not unprecedented for a single redundant tRNA to differ between taxonomically related species. For example, the tRNA pro found between tRNA Iso and control region 2 in C. horridus and A. piscivorus is not consistently present in all snake mtDNA ( Jiang et al. 2007). Other features include four genes with possible translation anomalies. The gene nad2 lacks a clear start codon. cox3 and nad5 have single-base insertions, and cob has a two-base insertion that would create frameshifts. All the anomalies were found in multiple runs (ve or more repeats from more than one clone). These could be the result of mitochondrial heteroplasmy in the individual and/or tissue that we sampled. It is also possible that these are the actual coding regions, in which case ribosome slippage (Farabaugh and Bjork 1999) could produce functional protein.

Acknowledgements Reagents were provided by Middle Tennessee State Universitys Department of Biology. Declaration of interest: The Applied Biosystems Genetic Analyzer 3130xl used for sequencing was purchased with a US National Science Foundation major instrumentation grant awarded to A.B.C. The authors report no conicts of interest. The authors alone are responsible for the content and writing of the paper.

References
Dong S, Kumazawa Y. 2005. Complete mitochondrial DNA sequences of six snakes: phylogenetic relationships and molecular evolution of genomic features. J Mol Evol 61: 1222. Farabaugh PJ, Bjork GR. 1999. How translational accuracy inuences reading frame maintenance. Embo J 18: 14271434. Gissi C, Iannelli F, Pesole G. 2008. Evolution of the mitochondrial genome of Metazoa as exemplied by comparison of congeneric species. Heredity 101:301 320.

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Jiang ZJ, Castoe TA, Austin CC, Burbrink FT, Herron MD, McGuire JA, Parkinson CL, Pollock DD. 2007. Comparative mitochondrial genomics of snakes: Extraordinary substitution rate dynamics and functionality of the duplicate control region. BMC Evol Biol 7:114. Lowe TM, Eddy SR. 1997. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucl Acids Res 25:955964.

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