pubmed article of crotalus horridus

3
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 (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 51 0 N, 868 18 0 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 blomhoffi (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 protein- coding genes were located and/or confirmed using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) and Virtual Ribosome (Wernersson 2006). tRNAs were initially identified using DOGMA and tRNAscan-SE (Lowe and Eddy 1997) and checked individually using Sequencher’s alignment features or by predicting secondary structure using mFold (Zuker 2003). Other mitochondria-specific features such as control regions and origin of light-strand replication were found using existing snake mitochon- drial genomes and Sequencher’s 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 ISSN 1940-1736 print/ISSN 1940-1744 online q 2012 Informa UK, Ltd. DOI: 10.3109/19401736.2012.722999 *Current address: Center for Human Genetics Research, 519 Light Hall, Vanderbilt University, Nashville, TN 37232, USA. Correspondence: A. Bruce Cahoon, Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA. Tel: þ 615 494 8792. Fax: þ 615 898 5093. E-mail: [email protected] Mitochondrial DNA, 2013; 24(2): 94–96 Mitochondrial DNA Downloaded from informahealthcare.com by University of Bristol on 12/09/13 For personal use only.

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Page 1: Pubmed Article of Crotalus Horridus

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

(Received 2 August 2012; accepted 17 August 2012)

AbstractThe complete mitogenome of the timber rattlesnake (Crotalus horridus) was completed using Sanger sequencing. It is17,260 bp with 13 protein-coding genes, 21 tRNAs, two rRNAs and two control regions. Gene synteny is consistent with othersnakes with the exception of a missing redundant tRNA Ser. This mitogenome should prove to be a useful addition of awell-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 510N, 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 blomhoffi (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 protein-

coding genes were located and/or confirmed using

Sequencher (Gene Codes Corporation, Ann Arbor,

MI, USA) and Virtual Ribosome (Wernersson 2006).

tRNAs were initially identified using DOGMA and

tRNAscan-SE (Lowe and Eddy 1997) and checked

individually using Sequencher’s alignment features

or by predicting secondary structure using mFold

(Zuker 2003). Other mitochondria-specific features

such as control regions and origin of light-strand

replication were found using existing snake mitochon-

drial genomes and Sequencher’s 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

ISSN 1940-1736 print/ISSN 1940-1744 online q 2012 Informa UK, Ltd.

DOI: 10.3109/19401736.2012.722999

*Current address: Center for Human Genetics Research, 519 Light Hall, Vanderbilt University, Nashville, TN 37232, USA.

Correspondence: A. Bruce Cahoon, Department of Biology, Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA.Tel: þ 615 494 8792. Fax: þ 615 898 5093. E-mail: [email protected]

Mitochondrial DNA, 2013; 24(2): 94–96

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Page 2: Pubmed Article of Crotalus Horridus

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 (five 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

University’s 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 conflicts 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:

12–22.

Farabaugh PJ, Bjork GR. 1999. How translational accuracy

influences reading frame maintenance. Embo J 18:

1427–1434.

Gissi C, Iannelli F, Pesole G. 2008. Evolution of the mitochon-

drial genome of Metazoa as exemplified by comparison of

congeneric species. Heredity 101:301–320.

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.

Mitogenome of timber rattlesnake 95

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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 7:1–14.

Lowe TM, Eddy SR. 1997. tRNAscan-SE: A program for improved

detection of transfer RNA genes in genomic sequence. Nucl

Acids Res 25:955–964.

Wernersson R. 2006. Virtual Ribosome–a comprehensive DNA

translation tool with support for integration of sequence feature

annotation. Nucl Acids Res 34:W385–W388.

Wyman SK, Jansen RK, Boore JL. 2004. Automatic annotation

of organellar genomes with DOGMA. Bioinformatics 20:

3252–3255.

Zuker M. 2003. Mfold web server for nucleic acid folding and

hybridization prediction. Nucl Acids Res 31:3406–3415.

J.B. Hall et al.96

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