Indian Journal of Biotechnology

Vol 15, October 2016, pp 495-506

Phylogenetic characterization of novel cathelicidin from Indian water buffalo

Shahid Hussain1, Chandra Sekhar Mukhopadhyay

1*, B V Sunil Kumar

1 and Simarjeet Kaur

2

1School of Animal Biotechnology, Post Graduate Institute of Veterinary Education and Research and 2Department of Animal Genetics and Breeding, College of Veterinary Sciences

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004, India

Received 5 May 2015; revised 17 November 2015; accepted 24 November 2015

The bubaline cathelicidins have been an important area of research because of possibility of exploring novel

antimicrobial peptides (AMPs) in the buffaloes, which are comparatively more sturdy animals and are better adapted to

Indian climate as compared to the crossbred cattle. The rationale behind the current study was to in silico characterize the

cloned bubaline cathelicidin3 (Cath3) peptide and to study the evolution of bubaline caths. Multiple sequence alignment of

the bubaline Cath3 (from this study) and homologous peptide sequences revealed an insertion of 6 amino acids in the

cathelin domain of the bubaline Cath3 peptide when compared with that of cattle. Biocomputational analyses of the Cath3

coding sequence (cds) as well as the amino acid sequences (using MEGA6 software & Datamonkey server) indicated that

different types and transcript variants of cathelicidin varied considerably within the same species, indicating the role of

natural selection during the evolution of caths. The cathelicidin cds belonging to divergent species were analyzed using

different models like SLAC, FEL and REL (Datamonkey server). It is concluded from the REL model that bubaline

cathelicidin3 antimicrobial peptides have undergone episodic positive selection (in upto 36 codons) conferring selective

advantage in evolution of the peptide.

Keywords: Buffalo, cathelicidin, evolution, immunity, selection

Introduction The role of antimicrobial peptides (AMPs) in the

immune system of eukaryotes has opened up new

research areas directed towards their possible

exploitation in improving disease resistance in

domestic animals and humans. The immune system is

persistently evolving among species, given its

necessity to act against the unceasing evolution of

pathogenic microbes for acquiring resistance. Genes

functioning in host-pathogen interaction may be the

targets of directional or balancing selection more

often than the genes which are no way related to the

health and immunity of the living being1,2

.

AMP family is widely distributed among

eukaryotes3,4

. These AMPs are evolutionarily ancient

component of the innate immune system and are the

fundamental defensive weapons of multicellular

organisms5. AMPs show diversity in both ways, i.e.,

inter species diversity vis-à-vis within species

variations among higher organism in their spectrum of

activity and structure6. Mammals have two major

categories of AMPs, defensins and cathelicidins. The

precursors of cathelicidins are divided into a highly

conserved N-terminal pre-proregion, which includes

the conserved signal sequence and the cathelin domain

as well as the divergent C-terminal antimicrobial

domain. Proteolytic cleavage of the peptide at a stretch

of specific amino acid residues by specific processing

enzymes releases the antimicrobial domain from the

whole peptide7-9

. Antimicrobial nature of these peptides

is attributed to their positive charge and their ability to

fold into amphipathic structure around a lipid bilayer

membrane10

.

The emergence of multiple drug resistance has led to

the need for identifying novel and effective antimicrobial

agents. Given the wide continuum of activity of naturally

occurring AMPs towards different pathogens including

multi-drug resistant bacteria and fungi, these peptides are

being studied as new anti-microbial agents11

. The

indigenous buffaloes are known for disease tolerance as

compared to crossbred cattle12

. The current study could

give an insight towards the use of cathelicidin in the

development of novel antimicrobial drugs. Presently, few

cathelicidins of bubaline origin have been identified and

no work on its evolution with respect to their Bovidae

counterparts has been reported. The present study aims to

unveil the evolutionary perspective of cathelicidin

peptides of the bubaline species.

___________

*Author for correspondence:

Mobile: +91-161-2414023

csmbiotech@gmail.com

INDIAN J BIOTECHNOL, OCTOBER 2016

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Materials and Methods Peripheral blood samples were aseptically collected

in sterile, 50 mL centrifuge tubes using 0.5 M EDTA as

an anticoagulant. Blood samples were collected from

adult dairy buffalo housed at the Dairy Farm,

Department of Animal Genetics and Breeding, Guru

Angad Dev Veterinary and Animal Sciences University,

Ludhiana, Punjab, India. The experiment was approved

by the Institutional Animal Ethics Committee.

Isolation of total RNA

Peripheral blood mononuclear cells (PBMCs) were

extracted from whole blood using HiSep based gradient

centrifugation. The isolated PBMCs were resuspended in

1 mL of Trizol reagent (Invitrogen, USA) and the total

RNA was isolated from the PBMCs using 1× RNA-lysis

buffer (ammonium chloride, 0.83 g; ammonium

bicarbonate, 0.1 g; EDTA 0.5 M, 20 µL and DEPC

treated water (0.1%) up to 100 mL). The concentration

and integrity of total RNA was checked by Nanodrop

spectrophotometer (Thermo Scientific, USA). Total RNA

samples having absorbance ratio (260/280) between

2.0 and 2.1 were subjected to first strand cDNA synthesis,

using RevertAid cDNA synthesis Kit (Fermentas, USA)

according to manufacturer’s instruction.

Cloning of Cathelicidin Coding Sequence

The primers targeting cathelicidin3 coding

sequence (cds) were designed from published

nucleotide sequence of taurine cathelicidin3 (Cath3)

mRNA (NCBI acc. no. NM_174001.1). Two pairs of

the primers were designed from overlapping

fragments using online primer designing tool Primer3

(CAT3F: 5′-GACCATGGAGACCCAGAGG-3′,

CAT3R: 5′-CCAGGAGGCGGTAGAGATTA-3′;

Cat1F: 5′-TGAGCGGTCCTCAGAAGCTA-3′;

Cat1R: 5′-CCTTGATGTTGGCGTTCCCA-3′). PCR

was carried out in a final volume of 100 µL

[containing cDNA 400 ng, primer pairs 0.4 µM each,

1× PCR-buffer, MgCl2 25 mM, 2 units Taq DNA

polymerase (Invitrogen, USA)] in a thermal cycler

(ABI, USA). The annealing temperature of 53°C and

55°C were standardized for CAT1 and CAT3 primer-

pairs, respectively. The amplicon was checked for

quality by horizontal agarose gel (2%)

electrophoresis. The purified PCR products were

ligated into pGEMT easy cloning vector (Promega,

USA) and transformed into Escherichia coli TOP10

strain as per standard protocol given in by Sambrook

and Russel13

. Recombinant clones were screened

according to blue-white selection method on

Luria-Bertani (LB) agar plate containing ampicillin

(100 µg/mL) + X-gal (40 mg/mL) + IPTG (100 mM).

Recombinant white colonies were picked up, cultured in

LB broth and subjected to plasmid isolation13

using the

GeneJET™ Plasmid Miniprep Kit (Thermo Scientific,

USA). The presence of insert was confirmed by

restriction endonuclease digestion, using EcoRI. The

isolated plasmids were also confirmed by custom

sequencing from the Department of Biochemistry,

University of Delhi, New Delhi, India.

Sequence Analysis

Processing and Submission of Sequence Data

The electropherogram of the cds (forward and

reverse) of cathelicidin were displayed, quality

checked and analyzed using BioEdit sequence

alignment editor, Version 7.2.314

. The open reading

frame was deduced and two overlapping sequences

(421 & 169 bp nucleotides) were merged to obtain a

single complete coding sequence (612 bp), which was

submitted to DDBJ, Japan (http://getentry.ddbj.nig.

ac.jp/getentry/na/AB918736/) (Getentry Acc. No.

AB918736.2). The open reading frame of Cath3 was

translated in silico using Expasy “Translate” tool

(http://web.expasy.org/translate/).

Blast Analyses

The final cDNA sequence was subjected to NCBI

BLASTn15

(http://blast.ncbi.nlm.nih.gov/) and a total

of 84 full length cathelicidin cds belonging to different

cathelicidin variants, from divergent species were

retrieved from GenBank. The nucleotide and

corresponding amino acid sequences of different

cathelicidin types and transcript variants were arranged

in FASTA format and maintained in separate text files.

Multiple Sequence Alignment (MSA)

The amino acid sequences belonging to cathelicidin

types of different species were aligned using Multiple

Alignment Fast Fourier Transform (MAFFT)

(http://www.ebi.ac.uk/Tools/msa/mafft/) online server.

The overview of the alignment of all the 83 peptide

sequences was obtained to depict the specific

conserved and variable regions of the whole peptide.

The bubaline cath variants as well as other ruminant

caths were also subjected to MSA for the conserved

pre-proregion and the variable AMP region separately

to study the extent of conservativeness of the sequence.

Phylogenetic Analysis

The deduced amino acid sequences of cathelicidin

variants of divergent species were first subjected to

HUSSAIN et al: EVOLUTIONARY ANALYSES OF BUBALINE CATHELICIDIN3

497

selection of best evolutionary model, followed by

multiple sequence alignment and construction of

phylogenetic tree using maximum likelihood (ML)

method16

(MEGA 6 software)17

, with 1000 bootstrap

resampling18

. The maximum likelihood method

utilizes optimality criterion to apply an explicit model

of evolution to phylogenetic tree construction16

.

Jones-Taylor-Thornton substitution model19

with

5 discrete Gamma categories and complete deletion of

missing data were selected as parameters. Tree

inference was drawn using nearest neighbor

interchange heuristic method. Evolutionary Divergence Estimation

The MEGA6 software17

was used for calculating

the evolutionary divergence (variance estimation by

100 bootstrap replications, Jones-Taylor-Thornton

substitution model19

, Gamma distribution with 5 rate

categories and homogeneity among lineages), amino

acid composition, disparity index to estimate the

homogeneity of substitution pattern (with complete

deletion of missing data and 100 Monte Carlo

replications) using the amino acid sequences. The

heatmap plots were generated using WGCNA

module20

of R program (Version 3.0.2) from the

matrix of evolutionary divergence.

Selection Pressure on Coding Sequences

The type of selection pressure operating on the cath

codons was determined using tests of neutrality

[Z-test of selection, using modified Nei-Gojobori

method (Jukes-Cantor)21

with transition/transversion

ratio of 2 and complete deletion of missing data] by

MEGA6 software17

. The codon based test of neutrality

compares synonymous and non-synonymous

substitution rates in protein-coding genes, and

considers a non-synonymous rate elevated above the

synonymous rate as evidence for Darwinian selection

for detecting adaptive molecular evolution22

. The

inferred number of synonymous (s) and

nonsynonymous (n) substitutions vis-à-vis the

estimated numbers of synonymous (S) and

nonsynonymous (N) sites were calculated using the

joint maximum likelihood reconstructions of ancestral

states according to a Muse-Gaut model23

of codon

substitution and Felsenstein 1981 model16

of

nucleotide substitution (data not shown). The variance

of the difference between the ‘N’ and ‘S’ was

computed with bootstrapping (500 replicates) using

the Nei-Gojobori method21

. The evolutionary

divergence between sequences was also estimated to

obtain the base substitution per site (using the

composite maximum likelihood method16

).

Analyzing Positive Selection Sites

A total of 33 complete cds were selected, which

were representing all the type variants of cathelicidin

of taurine, bubaline, ovine and caprine species and

one representative sequence each from wild Bactrian

camel, swine, killer whale, fresh-water dolphin and

minke whale (as outgroup). The specific codons that

have undergone positive selection were determined

by sequence analysis using Datamonkey

(www.datamonkey.org/) server24

applying the

statistical methods: Single Likelihood Ancestor

Counting (SLAC), Fixed Effects Likelihood (FEL)

and Random Effects Likelihood (REL)25

. At the

beginning, a model selection was run to test all of the

203 time-reversible models. A hierarchical testing

amalgamated with nested LRT tests with AIC

selection is done (http://www.datamonkey.org/help/

models.php) by the server to obtain a single "best-

fitting" rate matrix. The significance level was

separate for each of the models (SLAC, FEL &

REL)26

. Branch-site REL test26

for episodic

diversifying selection was done to undermine the

variation in the rate of evolution along both branches

and sites simultaneously (http://www.datamonkey.org/

help/index.php). This analysis enables us to determine

the lineages on which a subset of sites has evolved

under positive selection, without requiring prior

knowledge about which lineages are of interest.

Results and Discussion

Sequencing of Clones and Analysis

The clones of the overlapping partial sequences of

cathelcidin3, namely, CAT3 (169 bp) and CAT1 (463 bp)

(Fig. 1) were custom sequenced and the final

cathelicidin-3 (Cath3) cds of the Indian water buffalo

(Bubalus bubalis) was deduced. The functional

characterization of bubaline cathelicidin-3 has already

been done in our laboratory32

. Evolutionary Divergence of Antimicrobial Peptides

The lowest Bayesian information content (BIC)

score indicates the best model for analyzing the

substitution pattern of the coding sequences. In the

current study, the Jones-Taylor-Thornton (JTT)+

Gamma (G) was found to be the best model with the

lowest BIC score (8304.3). In this model, the Gamma

distribution (+G) adjusts the non-uniformity of

evolutionary rates among the sites of the codons19

.

INDIAN J BIOTECHNOL, OCTOBER 2016

498

MSA

MSA was done (using MAFFT27

) for divergent

species (represented as an overview of MSA in Fig. 2)

and also for bubaline cathelicidin types and transcript

variants (Figs 3 & 4) as well as different cathelicidin

types of the ruminant-species (Figs 5 & 6). The

pre-proregion of the full-length cathelicidin peptide is

conserved among the variants belonging to divergent

species, while the antimicrobial domain was highly

divergent among the cathelicidin types within same

vis-à-vis different species. The novel Cath3, obtained

from this study, presented an insertion of five amino

acids in the pre-proregion, which was absent in all

other ruminant species incorporated in the study (Fig. 5).

The structural annotations of the reported

cathelicidin peptides of divergent species and types

were collected from NCBI Protein database. The

functional domains of three divergent cathelicidin

peptides have been diagrammatically represented in

Fig. 7, which indicates that the length of the whole

cathelicidin peptide varies considerably among the

types within the same species. However, the length of

the signal peptide and the cathelicidin domain is

nearly same among the cathelicidin variants among

different species. There is much variation in the

mature peptide (harboring the AMP domain) among

different types of species-specific cathelicidin peptide.

Phylogenetic Analysis

The phylogenetic tree (Fig. 8) depicts that the

mammalian cathelicidins are clustering together,

while they are distinctly separate from that of the

aquatic and avian species. The branches representing

the Aves, Cetacea as well as the pseudoruminants

have been compressed to single nodes each, in order

to improve the resolution of the tree. Within the

mammalian species, the primates have formed a

separate clad from the non-primate, hoofed species.

This indicates that cathelicidin AMPs might have

undergone natural selection in accordance with the

requirement of environment and species-specific

needs to defend against microbes. The tree, in general,

demonstrates that different cathelicidin types

belonging to the same species have clustered in

different clads due to the difference in sequences.

Furthermore, some transcript variants (Cath5

transcript variants X1 and X2 of B. bubalis) fall in the

same node, while some (bubaline Cath4 transcript

variants X1, X2 & X3, X4) are not clustered within a

single node. Similar observations have also been

made with cathelicidin types and transcript variants of

other species like poultry with differences in

cathelicidin types of different breeds (not shown in

this phylogenetic tree). In certain cases, however,

different cathelicidin types from the same breed

(Fowlicidin1 and 3 from Brisson breed of chicken)

formed part of the same node. In porcine species,

some cathelicidin types like Protegrin2, 3 and also

Protegrin1, 4 formed part of the same node. The

cathelicidin types or variants belonging to the same

species present high bootstrapping values (>90% in

some cases), indicating the higher consistency of the

given data and taxonomical bipartitioning16

. Bootstrap

Fig. 1—Restriction digestion of CAT3 and CAT1 plasmids with restriction enzyme EcoR1 to release the inserts of defined sizes (169 & 463 bp).

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Fig. 3—Multiple sequence alignment (using MAFFT) of the conserved pre-proregion of different transcript variants of the bubaline cathelicidin variants.

Fig. 2—Depiction of the overview of the multiple sequence alignment (using MAFFT online server) of cathelicidin peptide sequences of all species under study.

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Fig. 4—Multiple sequence alignment (using MAFFT) of the variable AMP region of the transcript variants of the bubaline cathelicidin variants.

Fig. 5—Multiple sequence alignment (using MAFFT) of the conserved pre-proregion of ruminant cathelicidins.

HUSSAIN et al: EVOLUTIONARY ANALYSES OF BUBALINE CATHELICIDIN3

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values being indicative of the stability of branching

pattern, not the accuracy of the constructed tree.

Similar studies by Tomasinsig and Zanetti28

on the

evolution of bovine cathelicidins showed that most

sequences do not cluster monophyletically within

Bovidae, i.e., these sequences are more closely related

between than within species, suggesting the existence

of common ancestor genes before the divergence of

bovids from other cloven-footed animals like caprines

and ovines. Conversely, all porcine family genes

cluster monophyletically. This suggests that the

cathelicidin family genes presently observed in pigs

and bovids might have originated by independent

rounds of duplications and subsequent diversification

after the separation of Suidae and Bovidae28

.

Evolutionary Divergence of Cathelicidin Types and Transcript

Variants

The evolutionary divergence measures the departure of

sequences between two or more species with respect to

certain parameter. Here, we have compared the

evolutionary divergence of cathelicidins types and

transcript variants of different species. The analyses for

estimating the evolutionary divergence between amino

acid sequences were conducted using the Jones-Taylor-

Thornton matrix-based model18

and then Poisson’s

correction model, using MEGA6 software17

. The results

showed that the number of amino acid substitutions per

site was the minimum for a pair of sequences belonging

to the same clad, as is evident from the phylogenetic

tree. The evolutionary divergence estimates among

ruminant species like Pantholops hodgsonii (Tibetian

wild antelope), Ovis aries (sheep) and Bos taurus (cattle)

ranged 0.130-0.196. Gallus gallus (domestic chicken)

Fowlicidin and Tibetian antelope showed maximum

evolutionary divergence (heatmap plot; Fig. 9). The least

evolutionary divergent cathelicidin homologs belong to

the same families (dark blue). Intermediary evolutionary

divergence (light red, light blue & white) was noted in

case of porcine and equine cathelicidin types. A sample

dendrogram and heatmap of the evolutionary divergence

values among the cathelicidin peptide sequences of the

ruminant species was also generated for more clear

visualization of the evolutionary divergence (Fig. 10).

Ruminant cathelicidin transcript variants of same species

formed one node, indicating the minuscule variation of

evolutionary divergence. Furthermore, ruminant

cathelicidin variants were part of a larger clad implying

towards interspecies relatedness of ruminant

cathelicidins. In general, the sample dendrogram results

are comparable to that of evolutionary divergence

estimates.

Amino Acid Composition

The amino acid composition of bubaline

cathelicidin (Cath3) was compared with a closely related

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INDIAN J BIOTECHNOL, OCTOBER 2016

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Fig. 7—Comparison of cathelicidin domains among different cathelicidin types belonging to divergent species.

Fig. 8—Phylogenetic tree constructed from complete cathelcidin amino acid sequences using maximum likelihood with 1000 bootstrap resampling.

HUSSAIN et al: EVOLUTIONARY ANALYSES OF BUBALINE CATHELICIDIN3

503

Fig. 9—Evolutionary divergence heatmap: Relative distance among transcript variants vis-à-vis species specific cathelicidin.

Fig. 10—Sample dendrogram and evolutionary divergence of ruminant-specific cathelicidin types and transcript variants.

INDIAN J BIOTECHNOL, OCTOBER 2016

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species (taurine cattle, Cath4) and one distant species

(Chicken, Fowlicidin3). The differences in amino

acids among the cathelicidins of selected species have

been depicted in Fig. 11. Proline (17.09%) and

leucine (15.02%) were in maximum proportion in

bubaline cathelicidin3.

Homogeneity of Substitution Patterns between Sequences

Disparity index is used to measure the apparent

difference in evolutionary patterns for a pair of

sequences. A common assumption in comparative

sequence analysis is that the sequences have evolved

with the same pattern of nucleotide substitution

(homogeneity of the evolutionary process)29

. In our

study, disparity index (DI) was estimated for the

target amino acid of various species with respect to

that of the bubaline cathelicidin peptide (Cath3) and

was compared. Different cathelicidin types in cattle

and non-ruminant species, i.e., swine and equines

species, showed significant disparity indicating that

during evolution substitution pattern varied among

species with respect to buffalo. This implies that the

branching pattern obtained in the phylogenetic tree

could be misleading, as the assumption of

homogeneity of substitution pattern has not been met.

This limitation was further studied using “branch-site

REL analysis” (BSR)26

of the Datamonkey online

server (http://www.datamonkey.org/)25

. The BSR

method analyzes the episodic diversifying selection of

divergent homologous coding sequences. In this

analysis, in total 14 branches (or nodes) were detected

(p<0.05) to undergo episodic diversifying selection

(Fig. 12). This test estimates the ratio (ω) of

nonsynonymous (β) to synonymous (α) substitution

rates along with a lineage of interest, as a measure of

selective pressure. The branches of the tree in this

figure have different colours which signify the

strength of selection. Blue colour is indicative of

purifying selection (ω=0), black indicating neutral

selection (ω=1) and red colour implying

diversifying/positive selection (ω > 5). The variation

in thickness corresponds to the proportion of sites

undergoing harmonic diversifying selection. In this

case, three bubaline cathelicidins, namely,

XM_006065192|BbuCath6like|TVX1, XM_006065186|

BbuCath2like|TVX3 and XM_006040871|BbuCath

Fig. 11—Amino acid composition of cathelicidin peptides

belonging to buffalo (Cath3), taurine cow (Cath4) and chicken

(Fowlicidin3).

Fig. 12—Branch-site REL analysis of ruminant cathelicidin-types

and single, representative cathelcidin from other species. The red

oval shapes encircle the ruminant cathelicidins undergoing

episodic diversifying selection. The green coloured rectangle

indicates the bubaline Cath3 sequence of the present study.

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505

5like|TVX2, are shown to be undergoing harmonic

diversifying selection, with the taurine BC142015.1|

BtaCath5 also falling in this category. This suggests that

the ruminant cathelicidin sequences have been evolved

under heterogeneity of substitution pattern. Similar

statistical analysis has previously been attempted by

Singh and coworkers30

in the evolutionary analysis of

dicer.

Estimation of Selection Pressure for Various Codons

Maximum likelihood method is the codon

substitution model that was used to estimate the biases

introduced in nucleotide substitution and branch

length16

. Selection pressure on different codons of

cathelicidin types and variants was done using online

server Datamonkey (http://www. datamonkey.org/)25

.

Selection pressure on a codon was determined by

comparing the rates of synonymous (s) substitution per

synonymous site (dS) and non-synonymous (n)

substitution per non-synonymous site (dN). The value of

the test statistic dN-dS objectifies whether the codon

has undergone positive, negative or neutral selection.

Three different models, single likelihood ancestor

counting (SLAC), random effects likelihood (REL) and

fixed effects likelihood (FEL) were used to analyse26

the

data. SLAC yielded the minimum number of false

positive and false negative results, whereas REL model

detected more number of positively and negatively

selected codons compared to SLAC. In general, SLAC

is considered as the most conservative method, while

REL is the least intensive27-32

. SLAC model showed that

overall there were three positively selected codon sites

(23, 24 & 34), while REL showed as many as 36

positively selected codons (Figs 13A & B; Fig. 14). The

intermediary intensive FEL model showed 8 positively

selected codons (23, 24, 48, 50, 154, 162, 165 & 182).

The SLAC method for the cathelicidin sequences under

study presented somewhat uniform distribution of

positively selected codons based on the dN-dS values in

the conserved cathelin and the highly evolved

antimicrobial domain (Fig. 13A). On the other hand,

based on the REL method positive selected codons were

more dispersed towards the right of the graph, which

represented the antimicrobial domain (Fig. 14). The

REL method, in this case, gave a more factual picture of

positive selection of cathelicidin types and transcript

variants, as the incorporated input sequences were

divergent due to addition of the outgroup sequences in

the analysis.

The result from REL analysis clearly indicates that

almost all the positively selected sites are localized

within the highly variable, functional AMP domain of

cathelicidin. This signifies that the various

cathelicidin variants/types in the species under study

have a specific role to play in conferring immunity and

thereby fitness of the organism. Similarly, in buffalo,

although a very limited number of cathelicidin types

have been experimentally reported33

, the transcript

variants might have some specific roles. In a similar

type of work on ovine cathelicidin variants 1 and 2,

Dhaliwal et al34

reported that positive selection has

played an important role in the evolution of cathelicidin

variants among animal species.

Conclusion

Evidence of positive selection was seen in the

AMP, cathelicidin in the species that were included in

the study. The position of some of the positively

selected codons indicates that pathogens exert most of

Fig. 13 (A & B)—Graphical representation of the dN-dS test

statistic versus the codon positions obtained from SLAC (A) and

REL (B) analyses of cathelicidin cds.

Fig. 14—Positions of the positively selected codons of

cathelicidin peptides detected by REL analysis.

INDIAN J BIOTECHNOL, OCTOBER 2016

506

the selective pressures that lead to the changes observed.

Crystallographic studies would be helpful for assessing

the functional relevance of the cathelicidin.

Acknowledgement

The authors thankfully acknowledge the financial

assistance provided by the Department of Agriculture

and Cooperation, Ministry of Agriculture, Government

of India, New Delhi under Rashtriya Krishi Vikas

Yojana for conducting the present research work.

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