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Journal of Animal Science Advances
Molecular Genetic Analysis Based on Java Green Peacock (Pavo
Muticus) Mitochondrial D-Loop Efforts as a Basis for
Domestication in Probolinggo, East Java Indonesia
Aksono H. E. B. and Hermadi D. H. A.
J Anim Sci Adv 2014, 4(1): 668-674
Online version is available on: www.grjournals.com
AKSONO AND HERMADI.
ISSN: 2251-7219
Original Article
Molecular Genetic Analysis Based on Java Green
Peacock (Pavo Muticus) Mitochondrial D-Loop
Efforts as a Basis for Domestication in Probolinggo,
East Java Indonesia
1
1
Aksono H. E. B. and 2Hermadi D. H. A.
Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya-Indonesia.
2
Institute of Tropical Disease, Universitas Airlangga, Surabaya-Indonesia
Abstract
Besides hunting, habitate’s degradation and extinction were the main important factors lead to declination
of green peacocks population. Distance which separate populations in small island also become contact barrier
amongs the rest populations. These cause mating between close relatives which will lead to genetic downgrade
and local extinction. This study aimed to analyze whether peacocks from breeding cause changing in genetic
differences. Six green peacocks breeded from Probolinggo, East Java, Indonesia were being used in this study.
Its feather (calamus) were extracted for further examination. Polymerase Chain Reaction (PCR) technique with
D-loop mitochondrial-based primer was used to investigate the genetic differences of species amongs green
peacock. PCR products (330 bp) were analyzed using gel electrophoresis 1%, it were then purified and labeled
for the data sequencing. Sequencing data obtained reflects base order, which will be used to analyze genetic
difference amongs species (phylogenetic analysis) with the software program of Genetix Mac Ver 10.0. A green
peacock (Afropavo congensis) with the code of DQ 834 507 was being used as a reference. The results showed
that the feathers from the six green peacocks breeded in Probolinggo, East Java, Indonesia were genetically
different with green peacock which has been reported to GenBank with the code of DQ 834 507. Futhermore,
although amongs the six samples which were predicted to have close genetic relationship, but the code of MJM1; MJMJ-1; MJA-1; MJMA-1 were considered as young clusters / F2, while code MJD2 and MJD-1-1 were
considered as adult clusters / F1. Breeding can cause changing in genetic difference, which was showed in this
study that peacocks breeded in Probolinggo, East Java, Indonesia were genetically different compare to
reference DQ 834 507.
Keywords: Green birds peacock, PCR, mitochondrial d-loop, East Java-Indonesia.

Corresponding author: Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya-Indonesia.
Received on: 06 Nov 2013
Revised on: 29 Nov 2013
Accepted on: 08 Jan 2013
Online Published on: 26 Jan 2013
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J. Anim. Sci. Adv., 2014, 4(1): 668-674
MOLECULAR GENETIC ANALYSIS BASED ON …
Introduction
According to Delacour, 1977 quoted by by
Hernowo (2011) reported that in the world there are
two types of blue peacock (Pavo cristatus) spread
across India and Sri Lanka as well as the green
peacock (Pavo muticus) spread in Burma, Thailand,
Indochina, Malaysia, and Java.
Java green peacock (Pavo muticus muticus)
Linnaeus 1758, is currently only available in Java.
These birds are spread, with a population that is
relatively small in a variety of habitat types. Java
green peacock there in some kind of reserved area
status (nature reserves, wildlife sanctuaries and
national parks) as well as areas not reserved
(production forest, plantation) (Hernowo, 2011).
According Mackinnon (1988) quoted by by
Hernowo (2011) green peacock habitat consists of
forest and open area with a scrub is a favored spot.
Meanwhile Hernowo (1995), stated that the java
green peacock habitat is the monsoon forest,
lowland dry forest mixed with teak forests even
prairie. At this time the suspected habitat is capable
of supporting life is the green peacock nature
reserves, conservation areas and protected forests
and production forests of teak.
However, Kuroda (1936) quoted by by
Hernowo (2011) suspect that the green peacock
prefers dry areas in eastern Java. Problems related
to lives java green peacock among other high
hunting of peacock (eggs, feathers and individual),
java green peacock habitat destruction, narrowing,
and java green peacock habitat conversion. As a
result of the hunt can be lowered even wipe out
local populations java green peacock spreading in
some places. While it's still a lot of unknowns
related to parameters java green peacock population
of characteristics and even then the high pressure on
the population, but still java green peacock survive.
Van Balen et al., (1991) quoted by by Hernowo
(2011) stated that in the end of this decade,
poaching on java green peacock is the most serious
problem in causing endangerment peacock
population in java. Destruction, as well as a
narrowing conversion of forest would interfere with
peacock habitat both in quality and quantity of feed
components mainly, shelter and cover. With the
rampant land grabbing and illegal logging (1998 669
J. Anim. Sci. Adv., 2014, 4(1): 668-674
2003) will suppress or even eliminate the habitat
functions java green peacock.
Degradation and loss of habitat is a major
factor decrease pupolasi green peacock (Pavo
muticus), in addition to hunting factor (Holmes,
1989). Besides forest habitat located on a small
island separated by wide distances as well as the
relationship between the barrier remaining
population. Consequence may lead to fragmentation
of the peacock population. If this situation is
allowed to drag, in the future there will be a
marriage is feared that over time the close relatives
can lead to local extinction due to decreased genetic
quality (Hernowo, 2011).
The research and conservation efforts of the
green peacock (Pavo muticus) in Indonesia has not
been reported (Holmes, 1989), especially related to
molecular genetic research. reported population of
green peacock (Pavo muticus) in Indonesia in most
of Sumatra and Kalimantan and by IUCN reported
in the status of VU (vurnerable) are threatened with
extinction (McGowan dan Gasrson, 1995,
McGowan et al., 1995).
On the other side according to Sulandri and
Zein (2008), quoting from several sources say that
the domestication process led to some differences
and similarities between domesticated chickens and
red jungle fowl as his ancestors. therefore, through
this study wanted to find out whether the
conservation results of peacocks (domestication)
result in changes in genetic variation. It is very
important to keep known Indonesia superior germ
plasm can be sustained without changing the
genetic variation is mainly a decline in the quality
of genetic.
Therefore in the development of green peacock
(Pavo muticus), management factors necessary to
sustain a population through genetic enrichment
programs, where basic information can be identified
through phylogenetic reconstruction (Moritz, et al.,
1996). High genetic variability, that can resolve the
pressure due to changes in the environment (Avise,
1994; Hartl, 2000). Besides the high of
heterozygosity of a population is a good basic for
the development of subsequent populations.
molecular markers suitable for reconstructing
phylogenetic population is mitochondrial DNA
sequence variation. Excess of mitochondrial DNA
AKSONO AND HERMADI.
are: (1) High speed evolution of a relatively (5-10
times) when compared with nuclear DNA; (2)
Transmitted
through
the
maternal
line
intergenerational
without
undergoing
recombination, so that all molecule can be
considered as a single genetic unit that has many
alleles (Sudoyo, 1995); and (3) the relatively small
size so easily observed (Li and Graur, 1991;
Taberlet, 1996). In studying the closely allied types,
the use of nucleotide sequences of mtDNA control
region (D-Loop) can provide good resolution, this is
due to the mtDNA D-loop sequences containing
various mutations that have a rate 4-5 times faster
than other parts of the mtDNA (Horai et al., 1993).
Materials And Methods
Samples were taken from feathers (part of
calamus) of 6 green peacocks (Pavo muticus)
derived from breeding in Ponorogo, East JavaIndonesia.
The DNA was extracted from 25 mg (3-4 of
calamus) apex of the feather and added 180 µl Lysis
Buffer (100 ml of 1 Mol Tris, 200 ml of 0,5 M
EDTA, 2 ml of 5 M NaCl, 100 ml of 10 % SDS), 25
µl of 100 mg/ml DTT (dithiothreitol) and 20 µl of
10 mg/ml proteinase K, and than incubated at 500C
for 3-5 h until all of the feathers was melted. Added
400 µl phenol (Tris-HCl pH 8,0) an incubated for
30 min centrifuge at 13000 rpm for 3-5 min. Load
supernatant and place to other tube with 400 µl
chloroform isoamyl alcohol (24:1) and incubated
for 10 min. Than, centrifuge at 13,000 rpm for 3-5
min and take the pallet. Added ethanol 96% and 40
µl sodium acetat than incubated again at -200C for
45 min. Centrifuge again at 13,000 rpm for 30 min
and temperature 40C, wash with 10% ethanol and
centrifuge again at 13,000 rpm for 10 min and
temperature 40C. Disolve the DNA with 100 µl
TBE buffer (10 mM Tris-HCL pH 8.0, 1 mM
EDTA). Using spectrophotometer with wavelengths
260 and 280 nm for know the purified of DNA
(Sefc et al., 2003; Leeton et al., 1993; Malago et al.,
2002; Kimball et al., 1999).
PCR reaction use with primer GPDF (5’GGGGGGTATACTATGCATAATCGTG-3’) and
GPDR (5’-AAAGAATGGGCCTGAAGCTAGT3’), total volume for amplified is 25 µl, consists of
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1.5 µl of 10x PCR buffer (Sigma), 1.5 µl (25 mM
MgCl2), 100 ng each primer, 200 µM of each
dNTP, 0.5 U Taq DNA Polymerase, and 250 ng of
DNA. Condition of PCR amplification consists of
pre-denaturation 940C for 1 min 30 sec, 35 siklus
consists of 940C for 30 sec, 570C for 30 sec, and
720C for 1 min, than final extension at 720C for 10
min. PCR product is 330 bp (Wiwegwean and
Meckvichai, 2011).
A 330 bp PCR product visualized with agarose
gel containing 1% ethidium bromide (Sefc et al.,
2003; Leeton et al., 1993; Malago et al., 2002;
Kimball et al., 1999). Purified PCR products
obtained using methods such as the QIAGEN kit
(Wiwegwean and Meckvichai, 2011). After purified
do labelling sequenced using the ABI continued
with Prism 310 (Wiwegwean and Meckvichai,
2011). analysis of phylogenetic using sequence data
obtained mitochondria based D-loop and the
reference the data contained in GenBank with DQ
834507 code then processed using Genetix MAC
Ver 10.0.
Results and Discussion
Adult male java green peacock have straik crest
above the head and the cin have bluish, the long
hair was gold green and bronze green and usually
they have bigger size with body length around 210
cm (Sativaningsih, 2005: Hernowo 1995) (Figure
1). The purpose of this research to determine the
genetic diversity of mtDNA control a district has
been performed using PCR primers designed based
on mitochondrial D-loop region (Wiwegwean and
Meckvichai, 2011). Tthe sample is used part of the
distal feather (calamus) to expected that contains
many parts of the mitochondrial DNA. PCR results
using primer GPDF and GPDR designed from the
mitochondrial D-loop obtained a 330 bp on agarose
gel 1% and used for sequencing analysis (Figure 2).
Other than , this research want to determine the
genetic structure of the green peacock from
conservation and wild green peacocks including the
relation of genetic structure between F1 and F2. The
important thing to related of regulation that mother
of plants conservation and wild animals are
protected from natural habitats (W) expressed as a
state-owned and belong to the state. The mother
J. Anim. Sci. Adv., 2014, 4(1): 668-674
MOLECULAR GENETIC ANALYSIS BASED ON …
from conservation of wild animals first generation
(F1) and the captive from wild animals species
declared as a state-owned and belong to the state.
The mother from wild animals was protected from
natural habitats, and or the young of wild animals
from first generation (F1) has been protected, can
not be sold and must be submitted to the state.
Fig. 1: Java Green Peacock.
The results of this research showed that usually
green peacock from the conservation in East Java
has a different genetic structure with other peacock
has been reported in GenBank (DQ 834507). It is
suspected although one species because of the
distance and possible intermarriage between species
as well as the influence from conservation have
contributed to the formation of new species by
genetic structure (Figure 3 and 4).
F1 and F2 analysis in this research can not be
further explained due to the sample's identity is only
as a adults and young is not F1 related information.
However from the picture 5.9 shows that adult
green peacock form other clusters (MJD-1 and
MJD -2) while the young peacock green also set up
a cluster of other (MJM-1; MJMJ-1; MJA-1;
MJMA-1).
Green peacock females have the same color
pattern with peacock but soft, not bright, somewhat
dull and has no ornamental feathers. besides green
peacock females have a body length of 120 cm.
cover the top of the bronze-colored tail a greenish
with yellowish white color (Sativaningsih, 2005;
Hernowo, 1995; Hernowo 2011).
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J. Anim. Sci. Adv., 2014, 4(1): 668-674
330 bp
Fig. 2: PCR products of Green bird peacock (Pavo
muticus) isolates in East Java in the 1% gel
electrophoresis.
AKSONO AND HERMADI.
Green peacock have black spots duller brown
color. same color as the adult female but more
opaque. the chin and head covered with white
feather. crested start to grow after a two-week-old
young peacock. In general, the age of two months
younger peacock feather already has a perfect body
and a resembles adult female peacock but smaller
body size (Delacour, 1997 quoted by Hernowo,
2011).
With the decline in the population of peacock
green, various attempts have been made including
conservation efforts. According to Wiwegwean and
Meckvichai (2011) has performed research
specifically on Pavo muticus in Thailand related to
genetic variation arising from conservation. In this
case, the genetic diversity of conservation P.
muticus varies among populations depending on the
number and geographic reach. Differences
mitochondrial DNA (mtDNA) haplotype in a
population will demonstrate some of the origins of
the mother lineage. Thus, mtDNA variation
research will provide insight into the origin of the
female mother of the individual conservation results
and genetic structure of wild populations (not from
captive conservation). Moreover, it can provide
some evidence about the origin of the mtDNA
introgression via hybridization between P. muticus
and related species.
Fig. 3: One of Sequencing results of Green bird peacock (Pavo muticus) isolates in East Java.
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J. Anim. Sci. Adv., 2014, 4(1): 668-674
MOLECULAR GENETIC ANALYSIS BASED ON …
Fig. 4: Neighbour-joining phylogenetic tree of captive P. muticus based on the 330 nucleotide sequences of D-loop
gene from Green bird peacock (Pavo muticus) isolates in East Java.
According to tarwiningsih (2009), quoting from
several sources say that for the detection of genetic
diversity among the molecular basis using
mitochondrial DNA (mtDNA). This method is
widely used to study the genetic diversity of animal
and systematic relationships at various levels (Lamb
and Osentoski, 1995) due to the nature of maternal
mtDNA, which is purely derived from the female
mother. This method is widely used to study the
genetic diversity of animal and systematic
relationships at various levels (Lamb and Osentoski,
1995) due to the nature of maternal mtDNA, which
is purely derived from the female mother.
Mitochondrial genome also has a relatively small
size is ± 16,500 bp and has a rapid rate of evolution,
especially in the control (D-loop), giving rise to a
high diversity in mtDNA sequences intraspesies
(Aviase, 1994).
Genetic variation that appears in an organism is
the result of an evolutionary process, these
variations may occur due to a change in gene
frequency. According Tarwiningsih (2009) from
Elseth and Baumgerner (1984) said that there are
four factors that cause changes in gene frequencies,
there are natural selection, mutation, migration, and
abnormality of genetic. The natural selection is a
natural process in which some individuals have a
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J. Anim. Sci. Adv., 2014, 4(1): 668-674
genetic relationship to improve survival or
reproduction to adapt and have new generation that
can survive in the environment. The migration
causes individuals to move from one area to another
area. If the individuals survive and reproduce in the
new place, then they will inherit the genes in the
new environment. According Tarwiningsih (1984)
that the isolated population does not changes in
genes with neighboring groups. Many populations
are not fully insulated against other populations in
the same species, changes in the normal gene.
Conclusion
Breeding can cause changing in genetic
difference, which was showed in this study that
peacocks breeded in Probolinggo, East Java,
Indonesia were genetically different compare to
reference DQ 834 507.
Acknowledgement
This study was supported by a grant from
the Directorate General of Higher Education,
Departement of National Education, Indonesia,
funded 2013.
AKSONO AND HERMADI.
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