DNA Barcodes 2015; Volume 3: 1–4
Rapid Communication
Open Access
Rengarajan Rengasamy Lakshminarayanan, Achiraman Shanmugam, Archunan Govindaraju*
Sequencing of COI gene in four rodent pests for
species identification
Abstract: Among mammals, rodents are the most
important group since they influence the crop production
and protection. Species identification of rodents on
morphometric parameters is cumbersome and often
misleading. However, DNA based identification would
enable accurate species identification. For this purpose,
we examined 650 bp of Cytochrome c oxidase subunit1
(COI) in four rodent pest species namely Bandicota
indica, Milardia meltada, Rattus rattus and Tatera indica.
Our results show the maximum identity for Bandicota
indica and Rattus rattus within the species. For T. indica
the maximum similarity was observed with gerbillinae
species and M. meltada showed the maximum identity
with B. indica. The result indicates that COI based
identification can serve as an accurate and reliable tool in
the identification of rodent pest species.
Keywords: Species identification, COI gene, Rodent pest,
DNA Barcode, BLAST
Doi: 10.1515/dna-2015-0001
received September 16, 2014 accepted November 17, 2014
1 Introduction
Mammals are the most studied animal groups in species
identification. The taxonomy and species diversity
of mammals well documented [1]. Rodents constitute
the largest group of living mammal’s representing
about, 42% of world’s mammalian biodiversity [2].
*Corresponding author: Archunan Govindaraju: Center for
Pheromone Technology, Department of Animal Science,
Bharathidasan University,Tiruchirappalli-620 024, India, E-mail:
[email protected]
Rengarajan Rengasamy Lakshminarayanan: Center for Pheromone Technology, Department of Animal Science, Bharathidasan
University,Tiruchirappalli-620 024, India
Achiraman Shanmugam: Department of Environmental Biotechnology, Bharathidasan University,Tiruchirappalli-620 024, India
In Indian agriculture, rodents are one of the most
important vertebrate pests which are directly related to
the production and storage of crops [3]. In India more
than 18 rodent species are considered as commensal
and agriculture pests; among these Bandicota indica,
Millardia meltada, Rattus rattus and Tatera indica are
the most predominant pest species in both dry and
wet land agriculture and enjoy a wide distribution
throughout the country [4]. Many rodents also serve as a
reservoir for parasites causing diseases. Morphological
changes in Muridae family have been well reported
[5]. Identification of many small rodent species is
often difficult, considerably because of the wide
morphological variation throughout the development
during different life stages [1]. The intrinsic drawback
in morphology-based identification and the death of
classical taxonomists necessitates a new approach
to species identification [6]. Species-specific odour
plays a significant role in social communication in
mammals [7]; in order to make use this cue to develop
a pheromonal trap for a particular species individual
identification is important in rodent pest control.
Even though the sequences of animal mitochondrial
DNA are known to evolve quickly, however the gene
arrangements are not altered over a long evolutionary
time [8]. The 650 bp of mitochondrial cytochrome c
oxidase subunit 1(COI), is used as a standard species
marker for species identification in animal taxa [9,10].
In species identification the central concept is to match
the sequence of the confirmation entry to a reference
sequence through DNA sequence similarity searches.
Species identification of important rodent pests in India,
through COI gene sequencing is not well described. In
this study, we analyzed the 650 bp of cytochrome c
oxidase subunit 1 in four different rodent pest species
namely Bandicota indica, Milardia meltada, Rattus
rattus and Tatera indica.
© 2015 Rengarajan Rengasamy Lakshminarayanan, et al., licensee De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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2 R.R. Lakshminarayanan et al.
2 Materials and methods
3 Results and Discussion
Experimental animals were captured from agriculture
fields in various places of Cauvery delta region Tamil
Nadu, South India. We used burrow identification and
net trapping method for capturing Tatera indica and
Milardia meltada [11,12]; Bandicota indica and Rattus
rattus were trapped using steel and wooden traps [13].
Captured animals were incarcerated in polypropylene and
steel cages and transported to Bharathidasan University
animal house. Animals were identified up to species level
by expert taxonomist by using morphological characters
before further experiments [14]. Liver tissue were removed,
labeled and immediately stored at -80°C. Total DNA was
extracted from the stored tissue samples using standard
phenol/chloroform method with little modification [15].
The purity and concentration of the DNA
was
measured
spectrophotometrically
and
electrophoretically. For the amplification of 650bp of
COI genes the following forward and reverse primers
were used: 5′-CCTACTCRGCCATTTTACCTATG-3′ and
5′-ACTTCTGGGTGTCCAAAGAATCA-3′ [16]. The PCR
amplification reactions contained TrisHCl 10 mM ( pH
8.3); KCl 50 mM; forward and reverse primers at 0.5µM
each; 0.15 mM of dNTPs; 0.5 U of Taq polymerase and
with 1µL of DNA template. Thermocycling was carried out
in 96 wells Master gradient thermal cycler with the final
volume of 25µl. The PCR thermal cycler condition for the
amplifications was one initial denaturation step at 94°C for
2 min; 35 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1
min and an extension step of 72°C for 5 min. The amplified
products were verified on 1% ethidium bromide stained
agarose gels using a commercial (Genei, Bangalore) low
mass ladder for comparison. The PCR products were
eluted from the gel and purified using (RBC Gel elution kit,
Taiwan) kit according to the manufacturer’s instructions.
Sequencing was commercially done at MWG, India.
Sequences were then aligned (Table 1) by using BioEdit
[17]. The homologies of the obtained COI sequences were
blasted in National Center for Biotechnology Information
program (http://www.ncbi.nlm.nih.gov/BLAST/). The
sequence was entered in the Barcode of Life Data base
(BOLD, www.barcodinglife.org) with detailed specimen
information. All the four examined sequences were
submitted to GenBank (Accession numbers: JQ937326,
JN585971, JN547792, JN411131).
Among the data set of 650 bp of COI gene, 156 bp were
variable and 30 bp were parsimony informative. The
average proportions of T: C: A: G was 29.5: 25.8: 28.4:
16.3. The average sequence divergence among the four
species for COI gene was 47%. There were no insertions,
deletions, nonsense, or stop codons in the COI sequences
from all the samples. The blast searches from NCBI
showed 99% identity for Bandicota indica and Rattus
rattus. Sequence from Tatera indica and Milardia meltada
showed 86% similarity with gerbillinae species and 96%
similarity with Bandicota indica respectively. There were
no other reference sequence of COI gene for Tatera indica
and Milardia meltada. The present study reports for the
first COI barcode sequence of Tatera indica and Milardia
meltada. When a sequence was not found in the gene
database, the species identity relies on the closest available
matching sequence or even distantly related taxa [18]. The
accuracy of species identification relies on the quality
and length of the sequence. Mitochondrial COI gene is
reported to be the most conserved gene in animals and
hence its amino acid sequence has distinct advantages for
taxonomic studies [19]. The efficacy of COI gene in species
identification has also been reported in nematodes [20],
reedbuck [21], and Lepidoptera [22]. Accuracy of species
identification is very important in the field of integrated
pest management to know the entire distribution and life
history of particular species in order to make effective
control over the particular pest species. It is reported DNA
barcoding adopting COI gene is useful in identification of
insect species at all developmental morphological stages
[23]. Therefore, our findings support the development of
reference barcode database for the correct identification
of rodent pest species through DNA based identification
which may serve as an additional conforming method
along with the initial morphological identification.
Acknowledgement:
We
thank
Dr.
Chellam
Balasundaram, Professor of Emeritus, Bharathidasan
University for critical reading of the manuscript. We
thank Bharathidasan University, Tiruchirappalli,
for the award of University Research Fellowship. We
gratefully acknowledge the facility availed through
UGC, DST and UGC-SAP Government of India.
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Sequencing of COI gene in four rodent pests for species identification 3
Table 1: Consensus sequence alignment of mitochondrial cyochrome c oxidase gene [COI] from adult species of Bandicota indica, Milardia
meltada, Rattus rattus and Tatera indica. All sequence analysed were 650 bp in size.
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4 R.R. Lakshminarayanan et al.
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