Genetic Identification of Ceriops decandra (Chiru Kandal) using tRNA (Leu) Molecular Marker
Ceriops decandra is a glabrous shrub belongs to a family Rhizophoraceae distributed in a region including Southeast India, East Africa and Australia. The present study aimed to identify a tool in identifying the mangrove at the molecular level. The chloroplast trnL region was amplified from extracted total genomic DNA using the Polymerase Chain Reaction (PCR) and sequenced. Sequence of the principal agarose gel band revealed that Ceriops decandra and deposited in NCBI with the accession No. JN871232.
Received: December 22, 2011;
Accepted: February 20, 2012;
Published: May 14, 2012
Wetland ecosystems are imperative part of the natural resource pedestal in
India, which includes salt marshes, mangroves, coral reefs, seaweed and sea
grass. Of these, mangroves are predominantly important in view of its central
role both from the ecological and economical development. The total area of
mangroves in India is estimated to be 6,740 km2 and in Tamil Nadu,
mangrove coverage is about 150 km2 (Sathya and
Sekar, 2012). Ceriops decandra (Griff.) Ding Hou is a mangrove shrub
belongs to the family Rhizophoraceae, growing in drier mud near the inland limits
of normal tides. It is originated in a region including East Africa to Australia
through Madagascar, India, Bangladesh, Sri Lanka, Burma, Thailand and Malaysia.
Recent research has shown that the range of C. decandra is restricted
to the east coast of India and Bangladesh, Southwestern Thailand and Western
part of the Malay Peninsula. Economically C. decandra barks has huge
amount of non-timber forest product of tannins widely used in the fishing nets,
leather and chemical industries (Datta et al., 2011).
Also it has therapeutically potential triterpenoids (Prasad
et al., 2011). The middle and Southern part of its range is now considered
to be C. zippeliana (Sheue et al., 2009).
Also few researchers reported anti-nociceptive, hepatitis, anti-diabetic and
anti-oxidant property of C. decandra (Uddin et
al., 2005; Magwa et al., 2006; Nabeel
et al., 2010; Krishnamoorthy et al., 2011).
Genetic analysis of mangroves is urgently necessary to combat biotic and abiotic
stress. Number of molecular markers such as randomly amplified polymorphic DNA,
Amplified fragment length polymorphism, Inter simple sequence repeat and simple
sequence repeat studies have been used mainly for genetic diversity analysis
of plants (Bandyopadhyay, 2011). Among these RAPD, RFLP,
Protein and ISSR markers had been used to study diversity of C. decandra
(Schwarzbachl and Ricklefs, 2001). Nevertheless, these
molecular markers are dominant and they do not permit the differentiation of
heterozygous from homozygous accessions. These traits make them extremely suitable
to study diversity in supposedly related populations. Hence, the present study
was taken up to identify a tool in identifying the medicinally potential mangrove
species Ceriops decandra at the genetic level using trnL marker.
MATERIALS AND METHODS
Plant material and DNA extraction: Fresh leaves of Ceriops decandra
were collected from Pichavaram mangrove forest (Lat 11.42, Log 79.79) Tamil
Nadu, India. The specimen was botanically certified and a voucher specimen (AUCASMB06)
deposited in the Herbarium of Centre of Advanced Study in Marine Biology, Faculty
of Marine Sciences, Annamalai University, India. Plant DNA was isolated by Cetyl
trimethylammonium bromide (CTAB) protocol with modification described by Gurudeeban
et al. (2011). The yield of DNA per gram of leaf tissue extracted
was measured using a UV Spectrophotometer (Perkin Elmer, USA) at 260 nm. DNA
concentration and purity was also determined by running the samples on 0.8%
Polymerase chain reaction and DNA sequencing: The chloroplast trnL-F
regions were amplified from extracted total genomic DNA using the Polymerase
Chain Reaction (PCR) method. The universal primers 1, 2, 3 and 4 (Helini Biomolecules,
Chennai) of Taberlet et al. (1991) were used
to amplify trnL:
||trnL 5-3: GGTTCAAGTCCCTCTATCCC
||3- 5: ATTTGAACTGGTGACACGAG
Polymerase chain reaction parameters were an initial denaturation of 2 min
at 97°C; 30 cycles of 94°C for 1 min, annealing at 48°C for 2 min
and elongation at 72°C for 1 min, followed by an elongation step of 72°C
for 5 min. EtBr (Ethidium bromide) stained agarose gel was visualized under
a transilluminator. The fragment of interest was excised with a clean razor
blade. After removing the excess liquid, the agarose fragment was placed in
the spin column. The tube was centrifuged at 5500 rpm for 40 sec for the elution
of DNA. The eluent was checked by running on an agarose gel and observed on
a transilluminator and the DNA fraction was subjected for sequencing using trnL-F
specific primers (Helini Biomolecules, Chennai). Sequencing reactions were carried
out with ABI PRISM Dye Terminator Cycle Sequence Ready Reaction Kit (Applied
Biosystems Inc., USA). The obtained sequence was compared to the sequences in
NCBI using the BLAST algorithm to search for close evolutionary relatives (Altschul
et al., 1997).
RESULTS AND DISCUSSION
The genomic DNA of the C. decandra was subjected for the isolation of the DNA coding for trnL (Fig. 1) by using Polymerase chain reaction. The bands were cut and eluted and the DNA so obtained was subjected for sequencing. The sequence analysis demonstrated that all the corresponding bands on agarose gel belonged to C. decandra. Upon sequencing of the amplified DNA, the data obtained corresponds to 550 bases for C. decandra and deposited in NCBI with the accession No. JN871232 (Fig. 2) and the folded structure of tRNA L sequence shown in Fig. 3.
Phylogenetic association of C. decandra: Sequence of the foremost
agarose gel bands revealed that C. decandra in tested leaves was 100%
similarity to the trn (Leu) sequences of C. decandra found in
NCBI Genome Databank. The phylogenetic tree was shown in Fig.
4. The parsimony informative of dataset consisted of 550 base-pairs with
manually aligned matrix. Gaps represents as binary characters, missing data
had no affect on the topology.
||Genomic DNA of the C. decandra, Lane-1: Lamda DNA,
Lane-2: Plant DNA
The 100% bootstrap consensus tree is shown in Fig. 2. The
trnL gene sequence was of C. decandra blasted against nr database
and the top hits were taken and aligned by multiple sequence alignment. The
picture shows the phylogenetically related sequences. Our sequence is the first
entry in Genbank, there was no homologous sequence for C. decandra in
India. The topology is consistent with the maximum parsimony tree, which is
more resolved within the clades of Rhizophoraceae.
The chloroplast DNA trnT-F region in land plants consist series of conserved
trn genes trnT (UGU), trnL (UAA) and trnF (GAA)
arranged in tandem and separated by noncoding spacer regions. The region is
positioned in the large single copy region, approximately 8 kb downstream of
rbcL (Besendahl et al., 2000). The apparent
absence of gene rearrangements in the trnT-F region made the design of
plant universal primers possible. As a result, the trnL-F region, comprising
the trnL intron and trnL-F spacer, has become one of the most
widely used chloroplast markers for phylogenetic analyses in plants (Quandt
et al., 2004). The increased number of trn sequences from
a wide range of plants has allowed further study of structures, functions and
evolution in diverse orders of flowering plants, in basal angiosperms, in land
plants (Bakker et al., 2000; Borsch
et al., 2003).
The combination of trnL sequences with chloroplast markers rbcL
and matK for phylogenetic reconstruction in the tropical flowering plants
reported morphological character evolution, classification (Richardson
et al., 2004), biogeography and molecular dating (Richardson
et al., 2004; Pirie et al., 2006;
Pirie and Zapata, 2004).
|| Partial chloroplast tRNA-Leu trnL gene sequence of
medicinal mangrove Ceriops decandra
|| Folded structure of tRNA-Leu trnL gene sequence of
These markers appeared to contain complementary phylogenetic signals, as is
expected from different sequences sampled from the plastid genome (Chase
and Cox, 1998) and were thus applied in combined analyses. The combined
analyses yielded better resolved phylogenies, subject to higher levels of support,
than those derived from individual markers.
||UPGMA method of phylogenetic analysis of trnL gene
sequence of C. decandra compared with Rhizophoraceae clades
The present study can be utilized in identifying the mangrove species directly by using any part of the tissue in live form or even from the fossil specimens. The gene sequences of trnL-F will be the same in any part of the plant because of the presence of chloroplast and its genome is same throughout. In India, there was no sequence available in the database for medicinal mangrove till now and hence it can be exploited for the identification of mangrove species up to the level of varieties as the sequences will be more or less conserved with minor variations.
The authors are gratefully acknowledged to the Director and Dean, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period.
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