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Li 2013
Li 2013
Li 2013
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ISSN: 1940-1736 (print), 1940-1744 (electronic)
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Northwest A&F University, Yangling, Shaanxi Province 712100, People’s Republic of China
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Abstract Keywords
To study the genetic relationships of Pomacea canaliculata collected from rice fields in China, Mitochondrial DNA, phylogenetic analysis,
the mitochondrial (mt) 12S and 16S of 9 P. canaliculata isolates from 5 southern provinces in Pomacea canaliculata, sequence variations
China were sequenced and analyzed. The intra-specific sequence variations of P. canaliculata
were 0–1.1% for 12S and 0–0.6% for 16S, while the inter-specific variations among common History
Pomacea species in mt 12S and 16S were 3.0–11.7% and 2.3–10.1%, respectively. Phylogenetic
analysis based on combined sequences of mt 12S and 16S revealed complex genetic structure Received 3 May 2013
of P. canaliculata in China. Two phylogenetic groups of P. canaliculata were indicated in China Revised 6 May 2013
with one group sistered to P. canaliculata isolates from USA, and two groups were even found Accepted 21 May 2013
in the same province. The phylogenetic relationships of Pomacea spp. also could be effectively Published online 18 July 2013
inferred by combined sequences of mt 12S and 16S. These findings provided basic information
For personal use only.
for further study of population genetics and diffusion pattern of P. canaliculata in China as well
as in the world.
Table 1. Geographical origins of Pomacea samples used in the present study, as well as their GenBankÔ accession numbers for portions of
mitochondrial 12S and 16S genes.
of primer Sequence (50 –30 ) temperature ( C) over which the two sequences are compared (Chilton et al., 1995).
For 12S The inter-species genetic differences among Pomacea spp. (Table 1)
12SF TTAAAACTYAAAGGRCWTGGCGG 48 were evaluated using Megalign procedure in DNASTAR 5.0
12SR TTACTTTYAAGTCCWCCTTC 48 (Burland, 2000). In addition, base composition, transition and
For 16S transversion were calculated by Mega 4.0 (Tamura et al., 2007).
16S1 CGCCTGTTTATCAAAAACAT 52 To determine the phylogenetic relationships of P. canaliculata
16S2 CTCCGGTTTGAACTCAGATC 52 in the present study, the phylogenetic trees were re-constructed
based on the combined sequence of mt 12S and 16S, with Marisa
planogyra (EF519165) as the outgroup. Ambiguously aligned
regions were excluded using Gblocks online server (http://
using spin column purification (WizardÕ SV Genomic DNA
molevol.cmima.csic.es/castresana/Gblocks_server.html). The
Purification System, Promega, Madison, WI) according to the
remaining sequences were aligned and used for phylogenetic
manufacturer’s recommendations.
analyses using the maximum parsimony (MP) and maximum
likelihood (ML) methods. The MP analyses were carried out
Enzymatic amplification and sequencing
using PAUP 4.0 Beta 10 program (Swofford, 2002), in which
Portions of 12S (p12S) and 16S (p16S) were amplified from each bootstrap value was calculated from 1000 bootstrap replicates.
gDNA sample with primers listed in Table 2, which were reported The ML analyses was performed using PhyML 3.0 (Guindon &
previously by Colgan et al. (2007) for p12S and Rawlings et al. Gascuel, 2003), and the GTR model with its parameters for the
(2007) for p16S. PCR reactions (25 ml) were applied using concatenated dataset. The bootstrap values for ML trees were
reaction system containing 3 mM of MgCl2, 2.5 mM of each calculated using 1000 bootstrap replicates. Phylograms were
primer, 0.2 mM of each dNTPs, 1.25 U of ExTaq DNA drawn using the Tree View program version 1.65 (Page, 1996).
polymerase (Takara), and 1 ml of each gDNA template. PCR
reactions were validated and performed using the following Results and discussion
protocol: 94 C for 5 min (initial denaturation), followed by 35
Genomic DNA samples were extracted from 9 P. canaliculata
cycles of 94 C for 45 s (denaturation), 48 C (for p12S) or 52 C
isolates from 5 different provinces in China. Amplicons of 12S
(for p16S) for 30 s (annealing), 72 C for 1 min(extension), and
and16S were amplified individually and appeared as single band
then 72 C for 10 min (final extension). Samples without gDNA as
in agarose gel electrophoresis, with approximately 300 bp (p12S)
negative control were also included. Each amplicon (4 ml) was
and 500 bp (p16S), respectively. No size variation was observed
examined on agarose gel (1%). Positive amplicons were sent to
on agarose gel for each amplicon examined (not shown).
Sangon Company (Shanghai, China) for sequencing.
Sequence variations in the p12S and p16S regions among 9
P. canaliculata isolates were examined after direct sequencing. The
Sequence analysis and re-construction of phylogenetic
AT contents were 67.29–68.42% and 67.84–68.60% in p12S and
relationships
p16S, respectively. The intra-specific variations in p12S and p16S
Portions of 12S and 16S rRNA were separately aligned using the were 0–1.1% and 0–0.4% for samples from Guangdong and Hainan
computer program Clustal X 1.83 (Thompson et al., 1997) and the province. The differences in p12S and p16S were 0.8–1.9% and 0–
ambiguous bases at both ends of these sequences were discarded. 0.4% between isolates from China and USA, respectively (Table 3).
DOI: 10.3109/19401736.2013.809452 Phylogenetic analysis of Pomacea canaliculata collected from rice fields 3
Among all samples from 5 provinces in China, the genetic members of Pomacea species available in GenBank were 3.0–
variations in p12S and p16S were 0–1.1% and 0–0.6%, respectively. 11.7% in p12S and 2.3–10.1% in p16S (Table 3).
Intra-specific nucleotide variations represented transitions (A $ G, or To further infer the genetic relationships of P. canaliculata
C $ T, n ¼ 3 for p12S, n ¼ 2 for p16S), transversions (T $ G, n ¼ 0 isolates collected from rice fields in China, the combined
for p12S, n ¼ 1 for p16S) and base deletions (n ¼ 0 for p12S and n ¼ 3 sequences of p12S and p16S with total length of 779 bp were
for p16S). And, the inter-species sequence differences among used for the phylogenetic analyses. Topologies of trees, recon-
Table 3. Pairwise comparison of sequence differences (%) in portions of mitochondrial 12S (above the diagonal) and 16S (below the diagonal) among
Pomacea species.
PCG1 PCG2 PCG3 PCT PCH1 PCH2 PCH3 PCY PCGX PC1 PC2 PI1 PI2 PP1 PP2 PD1 PD2 PH1 PH2
PCG1 – 1.1 1.1 0 1.1 1.1 0 0 1.1 0.8 0.8 3.4 3.4 11 10.6 11.3 11.3 11.7 11.7
PCG2 0.4 – 0 1.1 0 0 1.1 1.1 0 1.9 1.9 3.0 3.0 9.8 9.8 11.3 11.3 10.5 10.5
PCG3 0.4 0 – 1.1 0 0 1.1 1.1 0 1.9 1.9 3.0 3.0 9.8 9.8 11.3 11.3 10.5 10.5
PCT 0 0.4 – 1.1 1.1 0 0 1.1 0.8 0.8 3.4 3.4 11 10.6 11.3 11.3 11.7 11.7
PCH1 0.4 0 0 0.4 – 0 1.1 1.1 0 1.9 1.9 3.0 3.0 9.8 9.8 11.3 11.3 10.5 10.5
PCH2 0.4 0 0 0.4 0 – 1.1 1.1 0 1.9 1.9 3.0 3.0 9.8 9.8 11.3 11.3 10.5 10.5
PCH3 0 0.4 0.4 0 0.4 0.4 – 0 1.1 0.8 0.8 3.4 3.4 11 10.6 11.3 11.3 11.7 11.7
Mitochondrial DNA Downloaded from informahealthcare.com by University of Alabama on 07/30/13
PCY 0.2 0.6 0.6 0.2 0.6 0.6 0.2 – 1.1 0.8 0.8 3.4 3.4 11 10.6 11.3 11.3 11.7 11.7
PCGX 0.4 0 0 0.4 0 0 0.4 0.6 – 1.9 1.9 3.0 3.0 9.8 9.8 11.3 11.3 10.5 10.5
PC1 0 0.4 0.4 0 0.4 0.4 0 0.2 0.4 – 0 2.6 2.6 9.8 9.8 10.6 10.6 10.9 10.9
PC2 0 0.4 0.4 0 0.4 0.4 0 0.2 0.4 0 – 2.6 2.6 9.5 9.5 9.8 9.8 10.5 10.5
PI1 2.7 2.3 2.3 2.7 2.3 2.3 2.7 2.9 2.3 2.7 2.7 – 1.5 10.2 10.2 12.5 12.5 12.0 12.0
PI2 2.9 2.5 2.5 2.9 2.5 2.5 2.9 3.1 2.5 2.9 2.9 1.4 – 10.2 10.2 10.9 10.9 11.3 11.3
PP1 5.7 5.1 5.1 5.7 5.1 5.1 5.7 5.9 5.1 5.7 5.7 6.1 6.2 – 0 14.8 14.8 12.9 12.9
PP2 5.7 5.1 5.1 5.7 5.1 5.1 5.7 5.9 5.1 5.7 5.7 6.1 6.3 0 – 14.8 14.8 12.9 12.9
PD1 9.2 9.7 9.7 9.3 9.7 9.7 9.2 9.4 9.7 9.3 9.2 9.6 9.4 9.8 9.6 – 0 9.1 9.1
PD2 9.4 9.5 9.5 9.5 9.5 9.5 9.4 9.6 9.5 9.5 9.4 9.7 9.6 10.4 10.2 0.2 – 9.1 9.1
PH1 9.6 9.3 9.3 10.1 9.3 9.3 9.6 9.7 9.3 10.1 9.6 9.9 9.9 10.0 10.2 8.2 8.5 – 0
PH2 9.6 9.3 9.3 10.1 9.3 9.3 9.6 9.7 9.3 10.1 9.6 9.9 9.9 10.0 10.2 8.2 8.5 0 –
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Figure 1. Phylogenetic relationships of Pomacea canaliculata samples from different province in China, with other Pomacea spp. inferred by
maximum parsimony (MP), using the combined dataset (12S þ 16S), with Marisa planogyra as the outgroup. Bootstrap values (in percentage) above
50% are shown for maximum parsimony (the first value) and maximum likelihood analyses (the second value). Weak refers to nodes recovered in the
optimal parsimony or likelihood tree, but not found (550%) in the bootstrap majority-rule consensus tree.
4 X.-Y. Li et al. Mitochondrial DNA, Early Online: 1–5
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Mitochondrial DNA Downloaded from informahealthcare.com by University of Alabama on 07/30/13
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Declaration of interest Parasitology 92:53–71.
Shi PJ. (1995). The natural disasters, constructions works for disesater
This work was supported in part by grants from the Funds of Basic reduction and sustainable development of China. J Nat Res 10:267–78
Research Program (QN2012003). The authors report no conflicts of (in Chinese).
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