Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

Vol 2 No 2: 61-70

Polymorphism of Microsatellite Loci on Y Chromosome in Long-Tailed Macaque Populations in Bali Island, Indonesia Polimorfisme Lokus Mikrosatelit Kromosom Y pada Populasi Monyet Ekor Panjang di Pulau Bali, Indonesia I Nengah Wandia 1,2*, I Gede Soma1,3, I Gusti Agung Arta Putra1,4 1 Primate Research Center, Udayana University 2 Laboratory. of Veterinary Anatomy, Faculty of Vet. Medicine, Udayana University 3 Laboratory. of veterinary physiology, Faculty of Vet. Madice, Udayana University 4 Laboratory. of Nutrition & Feed Animal, Faculty of Animal Husbandry, Udayana University *corresponding author, email: [email protected] ABSTRAK Monyet ekor panjang tersebar di seluruh wilayah di Pulau Bali, tetapi, kini keberadaannya telah terpisah-pisah menjadi populasi lokal yang lebih kecil. Walupun demikian, laporan yang melukiskan modus fragmentasi dan struktur genetik populasi belum banyak ditemukan. Penelitian ini bertujuan untuk mengeksplorasi polimorfisme lokus mikrosatellite kromosom Y pada populasi monyet ekor panjang di Pulau Bali dengan menggunanakan tiga primer mikrosatelit manusia, yaitu DYS390, DYS391, dan DYS393. Sejumlah 99 sampel darah dikoleksi dari monyet ekor panjang jantan yang berasal dari 8 populasi (23 dari Pulaki, 11 dari Bedugul, 13 dari Mekori, 8 dari Sangeh, 6 dari Uluwatu, 11 dari Alas Kedaton, 11 dari Ubud, dan 16 dari Bukit Gumang). DNA total diekstraksi dengan QIAamp® Blood Mini Kit. Polimorfisme lokus mikrosatelit dideteksi menggunkan teknik PCR, dan variasi alel dipisahkan melalui elektroforesis pada 7% gel poliakrilamid. Hasil penelitian menunjukkan bahwa lokus DYS390 dan DYS393 adalah monomorfik (masing-masing memiliki satu alel). Lokus DYS391 mempunyai dua alel, karena itu, lokus ini adalah polimorfik. Dari hasil penelitian dapat disarankan bahwa lokus DYS391 dapat digunakan untuk studi variasi genetik populasi monyet ekor panjang jantan di Pulau Bali. Kata kunci: monyet ekor panjang, polimorfisme, lokus mikrosatelit, kromosom Y, Pulau Bali ABSTRACT The long tailed macaques inhabit throughout Bali Island, however, recently they have been fragmented into many smaller local populations. In spite of that, there are just few reports that describe the mode of fragmentation and the genetic structure of the population. This research aimed to explore the polymorphism of microsatellite loci on Y chromosome in long tailed macaque populations in Bali Island using three human microsatellite primers, namely DYS390, DYS391, and DYS393. A total of 99 blood samples were collected from male long tailed macaques originated from 8 populations (23 from Pulaki, 11 from Bedugul, 13 from Mekori, 8 from Sangeh, 6 from Uluwatu, 11 from Alas Kedaton, 11 from Ubud, and 16 from Bukit Gumang). Total DNA was extracted using QIAamp® Blood Mini Kit. The polymorphism of the microsatellite loci was detected using PCR technique, and allelic variations were separated through 7% poly acrylamide gel electrophoresis. The result of the research showed that DYS390 and 61

Wandia et al.

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

DYS393 loci were monomorphic (each had only one allele). The DYS391 locus had two alleles, therefore, this locus was polymorphic. It could be suggested that DYS391 locus could be used to study genetic variation in male long tailed macaque population in Bali Island. Key words: long tailed macaques, polymorphism, microsatellite locus, Y chromosome, Bali Island flow homogenizes the genetic variation

INTRODUCTION

inter population (Nozawa et al., 1996; Long

tailed

macaque

(Macaca

Hartl & Clark, 1997; Li, 1997). However,

fascicularis) is a nonhuman primate that

the male migration some time gives a

has distributed worldwide (Euday, 1980;

negative effect, that is, it intruduces a

Fooden, 1980; Napier and Napier, 1985).

pathogen agent to the new population or

Nearly all islands of Indonesia have been

group in which the pathogen agent can

dwelling by the macaques, and because of

cause an illness or contagious disease

their high adaptation potency, the animals

(Wandia, 2003).

can live in various habitats (Napier-

Many tourist destinations in Bali

Napier, 1985; Fooden, 1995; Rowe, 1996).

Island are also a habitat of the long tailed

In the island of Bali, the macaques have

macaques

dispersed almost the regions and taken

Kedaton, Uluwatu, Pulaki, and Bedugul.

place in primary and secondary forests,

The macaques in such location give a

riverine, and coastal area (Southern, 2002).

positive impact, so that, the locations have

The long tailed macaque is a female

been known as the monkey tourist

philopatry i.e. live in social group in which

destination. Many non genetic researches

females remain and breed in the group of

have been carried out in these long tailed

their birth. The females compose a nuclear

macaques (Loudon et al., 2006; Lissa et

members of the social group (Joly, 1985;

al., 2008; Lane et al., 2010;, Schilaci et

Napier-Napier,

Perwitasari-

al., 2010; Brotcorne et al., 2011; Fuentes

Farajallah et al., 2004). Different from the

et al., 2011; Suarjana & Asmara, 2012).

female, male macaques will not stay firmly

The research about genetic variation in

in natal group, but migrate to other group

long tailed macaque population in Bali has

during their growing up period. The male

been conducted by Kawamoto et al.

migration would be a genetic bridge for

(1994) using blood protein marker. Other

entering the source of genetic variation to

research

the new population or group. This gene

microsatellite

1985;

62

like

i.e.

Sangeh,

Ubud,

characterization locus

on

Alas

of

somatic

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

chromosome has been carried out by

Vol 2 No 2: 61-70

13 samples.

Wandia (2003, 2004a, 2004b), and Rell et

The total DNA was extracted using

al. (2013). However, there was no study

QIAamp® DNA Blood Kits according to

that exploring the genetic variation using

company protocol (Qiagen, 2007). Three

molecular marker on Y chromosome of the

microsatellite loci on Y chromosome were

long tailed macaques. This research aimed

examined

to characterize the microsatellite loci on Y

human microsatellite primer (DYS390,

chromosome of long tailed macaque

DYS391 and DYS393). The loci were

populations

amplified by polymerase chain reaction

in

the

Island

of

Bali,

their

tecnique,

polymorphism

and

each

using

Indonesia. The polymorphic information

(PCR)

reaction

of loci is very important before using them

contained 4 mM MgCl2; dNTPs each 0.16

as molecular marker for assessing the

mM; a pair of primers each 0.4 mM; 0.7 U

genetic variation within and between

DNA Polymerase; 1.25 l PCR buffer

populations.

(10x), and 1 l template DNA. Pure water was added to make the final volume of reaction 12.5 l (Hillis et al., 1996). PCR

MATERIALS AND METHOD

for

DYS390

locus

was:

Pre

PCR:

A total of ninety nine male long tailed

denaturation (94o C) for 3 minutes; PCR:

macaque blood samples was subjected for

denaturation (94o C) 35 seconds, annealing

DNA extraction. The blood samples were

(50o C) 35 seconds, and elongation (72o C)

collected in the year 2010-2011 by

35 seconds; post PCR: elongation (72o C)

research team of Primate Research Center

5 minutes. PCR was replicated 30 cycles

Udayana University. The macaques were

using Applied Biosystems 2720 Thermal

anaesthetized with the combination of

Cycler. The PCR for DYS391 and

Ketamin HCl (dose 10 mg/kg body

DYS393 was the same as those of

weight) and Xylazin with ratio 5:1, and the

DYS390 with the exception of annealing

drug was injected using blow pipe. These

temperature at 54o C. The PCR product

samples

local

was separated by 7% PAGE in 1x TBE

populations in Bali Island, namely Sangeh

buffer and run at 125 Volt for 90 minutes.

8 samples, Uluwatu 6 samples, Alas

The allelic variation was developed using

Kedaton 11 samples, Ubud 11 samples,

silver staining, and the length of allele was

Bukit Gumang 16 samples, Pulaki 23

measured by aligning the allelic band with

samples, Bedugul 11 samples, and Mekori

the bands of 100 base pairs ladder of

originated

from

eight

63

Wandia et al.

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

molecular marker. The polymorphism of

length of the alleles was 282 base pairs

loci was analyzed using Alrequin Ver 3.5

and 292 base pairs. This locus was

soft ware (Excoffier & Lischer, 2010).

polymorphic, however, the two alleles were distributed oddly.

The allele 292

(DYS391292) was found in all populations,

RESULTS AND DISCCUSIONS

in contrary, the allele 282 (DYS391282) Results

was just found in population of Pulaki.

Distribution of alleles and haplotypes

Two other loci (DYS390 and DYS393)

The results showed that two alleles

were monomorphic as long as these loci

were found in DYS391 locus which the

just had one allele (Table 1).

Table 1. Allele Distribution of the Microsatellite Loci on Y Chromosome of Long Tailed Macaque Populations in Bali Island Relative Frequencies of allele in sampling site No

Loci

Allele

Total

PL n=23

BD MK n=11 n=13

SG n=8

AK n=11

UW n=6

UB BG Bali n=11 n=16 n=99

1

DYS390 DYS390240

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

2

DYS391 DYS391292

0.91

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.98

DYS391282

0.09

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.02

DYS393 DYS393272

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

3

Mapping of the three loci in one individual of Y chromosome will compose the haplotype. Genetic analysis showed that there were two kinds of haplotype, in which, one of them was only distributed to population of Pulaki (Table 2).

Table 2. Haplotype distribution of Y Chromosome of Long Tailed Macaque Populations in Bali Island Relative frequencies of haplotype in population Haplotype

hp composition

1 2

Total

PL BD MK n=23 n=11 n=13

SG n=8

AK n=11

UW n=6

UB BG Bali n=11 n=16 n=99

hp240:292;272

0.91

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.98

hp240;282; 272

0.09

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.02

64

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

Vol 2 No 2: 61-70

Locus and Haplotype Diversities of Y Chromosome of Long Tailed Macaque Populations in Bali Island Locus and haplotype diversities of Y chromosome of long tailed macaque populations were very low, or even zero in most populations (Table 3). It has some thing to do with the monomorphic condition of the loci. Polymorphic condition of DYS391 locus in population of Pulaki has contributed to the value of haplotype diversity (hd=0.17).

Tabel 3 Locus and Haplotype Diversities of Y Chromosome of Long Tailed Macaque Populations in Bali Island Diversity in populations

Total

Source

PL BD MK n=23 n=11 n=13

SG n=8

AK n=11

UW n=6

UB BG Bali n=11 n=16 n=99

DYS390 locus

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

DYS391 locus

0.17

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.04

DYS393 locus

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Haplotype (hd)

0.17

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.04

replication and due to the effective number

Disccusions A Locus polymorphism level is

of the variation source is a fourth apart of

supplied by the number of genetic

those of the somatic chromosome, this

variation sources in a population. The

genetic marker will reach a fixation in

variation of mtDNA genetic marker comes

shorter evolutionary time in a population

from one source, mtDNA of the female

compare to the genetic marker of somatic

(Walace et al., 1999; Ingman et al., 2000;

chromosome. The resulth of this research

Bowels et al., 2007), likewise, the

exibited three allales of the four alleles

variation of a genetic marker of Y

found were fixed or nearly fixed in all

chromosome

populations. Nearly fixation state of those

just comes from

Y

chromosome beared by the male (Evans et

alleles may associate with

al., 2010; Rovie-Rian et al., 2013). As

recombination during replication and low

long as a genetic marker of Y chromosome

effective number of variation source as

does not undergo a recombination during

describes above. There was only one allel,

65

no DNA

Wandia et al.

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

DYS391282, had low frequency and just

populations in Bali Island was lower than

found in population of Pulaki (Tabel 1).

those

This may reflect a new mutation of allele,

populations in Lombok Island. The botlle

therefore, the male bearing the allele has

neck effect may decrease severely the total

not had enough chances to distribute the

number of the population members, and so

allele through mating with a female within

do the number of the male. While this

a population as well as migrating to other

preliminary

populations.

microsatellite loci on Y chromosome and

of

the

long

tailed

resarch

macaque

used

three

The low haplotype diversities are not

only one of them was polimorphic, the

only affected by the total number of

data may not express the real condition. It

geneticaly different male in population,

would

but also the number of loci used and the

individual

polimorphism of the loci (Perwitasari-

populations.

Farajallah et al., 2004). Low haplotype

pertaining to the evolutionary history of

diversities of Y chromosome may reflects

male macaques in Bali Island, a further

to a few genetically different male of long

research

taild macaques in wild in Bali Island. The

microsatellite loci on Y chromosome is

long tailed macaques in Bali Island were

needed. Moreover, a broaden sampling

originated from Java Island and they

sites

migrated during the two islands unite to be

including population in Java Isalnd and

a part of Sunda Selft (Eudey 1980; Fooden

Lombok

1995). However, there is no a clear

evolutionary history of male macaque in

information about the

of

South Archipelago of Indonesia. However,

migration events and the total number of

this research has succeeded to characterize

different populations had migrated. This

three microsatellite loci on Y chromosome

lack of information brings to the difficulty

at native populations of long tailed

to justify the male number of long tailed

macaque in Bali Island.

frequencies

be

weak both

(population)

Island

differentiating

within

To

using

in

and

better

a

understand

more

may

to

between

be

polimorfic

obligated

elucidate

the

macaques as the founder population in Bali Island in the past. Kawamoto et al.

CONCLUSION

(1984) stated that the long tailed macaques populations in Bali Island had suffered

Based on the result of the research, it

from a bottle neck effect. It was based on

can be concluded that each of DYS390 and

the

DYS393 loci has one allele, so, these two

blood protein data that the genetic

variation

of

long

tailed

macaque

loci are monomorphic in male long tailed 66

Jurnal Ilmu dan Kesehatan Hewan, Agustus 2014

Vol 2 No 2: 61-70

macaques in Bali Island. On the other

Indonesia. In Monkeys on the Edge.

hand, DYS391 locus that having two

Ecology and Management of Long

alleles is polymorphic. The locus of

Tailed Macaques and Their Interface

DYS391 could be used in the study of

with Humans. Editors M.D. Gumert,

genetic differentiation between male long

A.

tailed macaque populations in Bali Island,

Cambridge University Press, Pp 180-

but not the two others.

182.

Fuentes,

and

L.Jones-Engel.

Eudey AA. 1980. Pleistocene glacial phenomena and the evolution of Asian

ACKNOWLEDGEMENT

macaques. In The Macaques. Studies My thanks

to

the President

of

in Ecology, Behavior and Evolution.

Udayana University for grand approval on

Edited by D.G. Lindburg. :52-83.

this research with the Contract No:

Evans BJ, Pin L, Melnick DJ, Wright SI.

21.4/UN14/KU.03.04/2012, on the date of

2010.

May, 16 2012 and the Contract No:

macaque monkeys: Implications for

174.17/UN14.2/PNL.01.03.00/2013,

effective population size and dispersal

on

the date of May,16 2013.

Sex-linked

inheritance

in

to Sulawesi. Genetics 185: 923–937 ( July 2010). Copyright _ 2010 by the Genetics Society of America DOI:

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