Abstract
Three complete mitochondrial DNA (mtDNA) control regions (CRs) of Chelodina rugosa (Ogilby, 1890), Chelus fimbriata (Schneider, 1783), and Podocnemis unifilis (Troschel, 1848) were firstly determined using Long-PCR method and the length were 1,016 bp, 1,149 bp, and 985bp, respectively. Together with CRs of Pelomedusa subrufa (Bonnaterre, 1789) and nearly complete CR of Podocnemis expansa (Schweigger, 1812) obtained from GenBank, the structural and evolutionary characteristics of mtDNA CRs in pleurodiran turtle were analyzed in this study. We identified three functional domains (TAS, CD, and CSB domains) as well as their conservation sequences (TAS, CSB-F, and CSB-1) according to their homology to those of other turtles. Within the TAS domain, an interrupted poly-C stretch was found in C. rugosa, C. fimbriata, and P. subrufa, which also exists in the published mt DNA CRs of Chrysemys picta (Schneider, 1783), Trachemys scripta (Thunberg in Schoepff, 1792), and Trionyx triunguis (Forskål, 1775). The analysis of the origin for the poly-C sequences in TAS domain from six turtles suggested that the poly-C sequences are more related to "goose hairpin" in birds rather than CSB2 in CSB domain. In the CSB domain, CSB2 and CSB3, which were determined in CRs of Cryptodira, were absent in Pleurodira CRs, indicating the regulative mechanisms of transcription may be varied in both two suborders and the lack of CSB2 and CSB3 could be proposed as one of diagnostic characters between Pleurodira and Cryptodira at molecular level. As for CR of other cryptodiran turtles, variable number of tandem repeats (VNTRs) in the 3' end of the CRs was found in the five pleurodiran turtles. Interestingly, the long repeated motifs from each species could form stable stem-loop secondary structures, suggesting that the repeated sequences may play an important role in regulating replication of the mitochondrial genome in Pleurodiran, and the secondary structures of VNTRs may provide some potential information in phylogenetic inference.
Highlights
The mitochondrial genome is highly conserved and compact, usually including genes that code for 13 proteins, 22 tRNAs and two rRNA, as well as an important noncoding sequence in vertebrates
We identified three functional domains (TAS, conserved domain (CD), and conserved sequence block (CSB) domains) as well as their conservation sequences (TAS, CSB-F, and CSB-1) according to their homology to those of other turtles
In the CSB domain, CSB2 and CSB3, which were determined in control regions (CRs) of Cryptodira, were absent in Pleurodira CRs, indicating the regulative mechanisms of transcription may be varied in both two suborders and the lack of CSB2 and CSB3 could be proposed as one of diagnostic characters between Pleurodira and Cryptodira at molecular level
Summary
Specimens of C. rugosa, C. fimbriata, and P. unifilis were stored at the Anhui Normal University. Total genomic DNA was extracted from their muscles with the proteinase K method (SAMBROOK & RUSSELL 2001) and kept at -20°C for PCR amplification. The mitochondrial CR was amplified by long-PCR. The entire CR and 3 tRNAs (tRNAThr, tRNAPr°, and tRNAPhe) as well as partial Cyt b and 12S rRNA gene sequences were amplified in one single step using a pair of long-PCR primers: LCR-F: 5’-CTTCCTATTTGCCTATGCTATC-3’ LCR-R: 5’-TATTTTGGGCTCCTGGTGTA-3’. Long-PCR conditions were: one minute at 95°C, 30 cycles of 10 seconds at 98°C, five minutes at 55°C, followed by a final extension for 10 minutes at 72°C. PCR products were isolated using a Gel Extract Purification Kit (TaKaRa Co., Ltd, Dalian, China) after 1% agarose gel electrophoresis. The purified Products were sequenced with an ABI3730 automated sequencer
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