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This is to certify that

Dr. drh. Trini Susmiati, MP. has attended the International Seminar

BITING FLIES AS VECTORS OF TRYPANOSOMIASIS AND THE ROLE OF ONE HEALTH IN ANIMAL HEALTH

as

SPEAKER

organized in collaboration by the

FACULTY OF VETERINARY MEDICINE, GADJAH MADA UNIVERSITY GREASE Network and BioZoonoSEA PLATFORM

KASETSART UNIVERSITY, MAHIDOL UNIVERSITY, CIRAD and IRD

With the support of the French Ministry of Foreign Affairs on the 19th- 25th May 2014, at the

Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia

DR. MARC DESQUESNES CHAIR

WORKSHOPOC BioZoonoSEA, Thailand

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DR. D~~ PRASTOWO, MSi DEAN

FACULTY OF VETERINARY MEDICINE GADJAH MADA UNIVERSITY

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.... I"~''·'"""' · n·•-'" I REPUBLIQ_UE FRANYUSE

, MINJSff:R.E

' DE> I AFFAIRE$ E"TRANGERES

'

This is to certify that

Dr. drh. Trini Susmiati, MP. has attended the International Seminar

BITING FLIES AS VECTORS OF TRYPANOSOMIASIS AND THE ROLE OF ONE HEALTH IN ANIMAL HEALTH

as

SPEAKER

organized in collaboration by the

FACULTY OF VETERINARY MEDICINE, GADJAH MADA UNIVERSITY GREASE Network and BioZoonoSEA PLATFORM

KASETSART UNIVERSITY, MAHIDOL UNIVERSITY, CIRAD and IRD

With the support of the French Ministry of Foreign Affairs on the 19"'- 25"' May 2014, at the

Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia

(\ I ,

- ~/r= DR. MARC DESQUESNES

CHAIR WORKSHOPOC

BioZoonoSEA, Thailand

PRASTOWO, MSi DEAN

FACULTY OF VETERINARY MEDICINE GADJAH MADA UNIVERSITY

..

M1NISTERE DES

!AFFAIRES ETRANGEREs

International Seminar And Workshop

BITING ES AS VECTORS OF TRYPANOSOMES AI\ THE ROLE OF ONE HEALTH IN ANIMAL HEALTH

GENETIC DIVERSITY STUDY OF NATIVE AND KEDU CIDCKENS USING RAPD­PCRMETHODE

Trini Susmiati1, Rini Widayanti 1

, PamungkasBagusSatriyo2

1 DepartemenBiochemistry of Veterinary Medicine, Universitas Gadjah Mada;2 Student of Faculty of Veterinary Medicine Universitas Gadjah Mada

Jl. Fauna, No. 2, Karangmalang, Sleman, DIY HP: 08158941016, Email: n·inisusmiati@vahoo.com

ABSTRACT

Chicken is a local Indonesian germplasm that need to be explored its potential. Currently there are several clumps has specific characteristics and some has potential for commercial poultry and for tourism purposes. Ontilnow ,supplyingpoultrynative to Indonesiais still a problem, because of the unavailabilityof commercialstrainswiththe performancestandard, sothere is nocommercialbreedingspecialized in providinglocalpoultry.ldentification and characterization efforts of kedu chicken and local chicken is still needed, it is considered that this activity is·very important as well as it is useful for Indonesiangermplasm conservation, helping original Indonesian chicken breeding programs and helpthe programs for genetic resources conservation (genetic diversity) Indonesian chicken.

Effortsto preserve thenativeof Indonesianchicken,andthe power ofnatural selectionwill determine thesurvival ofoffspringtoreproductive processes. Whilethe selectionis madecan bechosenbased onexcellenceforhuman needs. This study usedlocal chickenandkeduchicken fromTemanggungCentral Java.Bloodwas takentoisolateDNAusingconventionalmethods, phenolchloroform(Sambrooket al, 1996).DNAmoleculeswereobtainedamplifiedby PCR-RAPD andresultdielectrophoresisusing3% agarose. Polymorphisms ofDNA bandselectrophoresisresultswere analyzedtoseethe geneticdiversitybetween localchickenandkedu chicken.The results of RAPD-PCR using primers 3, 4, 5, and 6 indicate the presence of genetic diversity within populations and among populations, but can not be used as genetic markers. The closest genetic distance is KeduBlirik to KeduMerah and KeduPutih; as well as the KeduMerah andKeduPutih, and the most distant is local chicken and KeduHitam.

Keywords: Kedu chicken, local chicken, PCR-RAPD.

135

INTRODUCTION

Indonesia has many high-potential local poultry for farm development. One type of local

chicken with a great potential is village chicken, and the local chicken population mostly located

in rural areas. Local chicken isindonesiangermplasm, it is still need to unearthed potentiaL

Currently there are several clumps that have specific characteristics and have potential for

commercial poultry. The existence and potential of local chickens can support communities in

rural economy. Nataamijaya( 2000) promoted, that there are 31 local Indonesian chicken clumps

which has a characteristic of them : Pelung chicken, Keduchicken, Nunukan . chicken,

Sedayuchicken, Sentul chicken, crow chicken and other.

One way to increase the genetic quality of keduchicken IS continuous selection by

choosing keduchicken which has a high potential advantage to be developed. The genetic

diversity is one of the basic to determine the level of success of selection value changes in a

population and can also be used in the determination of the origin of livestock. Genetic diversity

can be examined using the characters alleles of a particular derived locus from body fluids or

tissues such as blood, spleen, heart, liver and other organs. With the help of multivariate analysis,

it can be specified section or size of certain body which can be distinguished from the group or

clump of an animaL In Molecular, animal identification can be performed to determine the

genetic diversity studies and genetic distance. The usefulness of genetic distance is needed to

support breeding programs, especially in implementing a cross between clumps (crossbreeding).

Increasing productivity through crossbreeding should be performed by mating between distantly

related clumps in order to obtain positive heterosis effect. Therefore we need an increase in

genetic quality to keep it maintained its sustainability. Identification and characterization kedu

effort is still needed, this activity is considered important as well as useful for

136

lndonesiangermplasm, itis also useful in helping breeding programs. Beside that, genetic distance

is needed to assist in the conservation of genetic resources program of local chickens in

Indonesia. Fumihito et al., (I 994 and! 996 ), that the local Indonesian chickens originated from a

common ancestor that is Red Forest chicken.

Analysis of the molecular basis of genetic diversity was rapidly evolving in present

moment, using the techniques of DNA analysis. Several kinds of methods can be performed such

as : RAPD - PCR ( Random Amplified Polymorphism DNA - PCR ), RFLP ( Restriction

Fragment Length Polymorphism ), fingerprint analysis (finger printing), minisatellite ( VNTR I

variable number tandem repeat ), microsatellite ( STR I Short tandem repeat ) and mitochondrial

DNA. RAPD - PCR method is a combination of PCR techniques using primers with random

sequences for genomic DNA loci amplifiklasi purposes. Use of RAPD-PCR method in the

analysis of the chicken kinship is more efficient in terms of cost and processing time as well as

methods was performed fairly simple and more accurate. The purpose of this study is to assess

the genetic potential of kedu chicken andlokal chicken (ayamkampung) as seen from the

diversity of genomic DNA for gene mapping, and specific DNA fragments characteristic of

Indonesian local chickens. As well as assessing the difference between local chicken

(ayamkampung)and chicken kedu of DNA loci.

This study aimstoidentifY and characterizekedu chicken, preservation ofkedu chicken as

germplasm, breeding program and genetic resources conservation program of Indonesian native

chicken

137

METHODS

The sample used in this study is domestic chicken and kedu chicken obtained from

Temanggung,Central Java. PCR primers used for RAPD Are: RADP3= 5' GTA GAC CCG T 3.;

RAPD4=5· AAGAGC CCG T "; RAPD5=,. AACGCG CAA c'"; RAPD6=,. CCC GTCAGCA ,.

Isolation of DNA

Blood samples were taken from the chicken, then extracted by adding extraction buffer (

JOOmMTris - HCI, 20 mM EDTA, 1.4 M NaCJ, 2 % CTAB, I % mercapto - ethanol ).

Subsequently was incubated at 65°C, and then the samples were centrifuged at 7600 rpm speed.

Supernatant obtained was added a solution of chloroform Isoamylalchohol (CIAA), then

' centrifuged at I 0,000 rpm. The supernatant obtained was separated and added warm NaCJ,

inverted for 5 minutes, and added again CIAA. The supernatant was separated by centrifugation

at l 0,000 rpm and then added with cold absolute ethanol, and incubated at room temperature.

The solution was centrifuged again at 13,000 rpm speeds.' Pellet obtained was washed twice with

70% using alhohol, then dried pellets, and added TE buffer pH 8.

Amplification of DNA (PCR-RAPD)

Amplification was performed in conditions: 3 min at 95° C, primer annealing for 45

seconds at 37 - 42°C, elongation at 72°C for I minute, the third stage of the last cycle was

performed 39 times. Then proceed to the post- elongation stage for I 0 minutes at 72°C. DNA

amplification reaction was performed on a sample volume of 25 ul, with the following

composition : 8mm dNTP mix, PCR buffer !Ox, MgCb, Taq DNA polymerase, DNA template,

primers ( I 0 pmol ), and then add distilled water to a total volume of 25 ul. DNA amplification

products were visualized using agarose gel electrophoresis with a horizontal 3 %. Ethidium

138

bromide was add toagarose gel that capable of doing chelate with the DNA, so it can be seen

under ultraviolet light.

Analysis of data

Mapping of DNA and the position of the reference range of local chicken and kedu

chicken diversity based of DNA bands was analyzed by PCR - RAPD on 3 % agarose gel in the

the binarydata. Data kinship was analyzed with UPGMA method in the MEGA program version

5.

RESULTS AND DISCUSSION

This study uses 26 chickens, which consists of Cemani ( 5), KeduHitam( 5), KeduPutih (

5), KeduMerah ( 5), KeduBiirik ( 5 ) obtained from Temanggung , Central Java and Buras

chicken ( 5 ) are taken in the area Purworejo, Central Java. Total DNA isolation results are

visualized on a I % agarose gel is presented in Figure I.

DNA was isolated and then used as a mold for PCR- RAPD. There are 6 kinds of primer,

however, because the results of RAPD - PCR using primers 1 and 2 is not good enough, then the

results ofRAPD- PCR using primers 3, 4, 5 and 6 were subsequently used for the analysis of the

diversity of research chickens. Results of PCR - RAPD were electrophoresed on 3 % agarose gel

using a lOObp DNA ladder as molecular weight DNA pointer. Results ofPCR-RAPAD using 3,

4, 5 and 6 primers were respectively presented in Figure I a, I b, 2a, 2b, 3a, 3 b, 4a and 4b.

139

(a)

8 Nov 2011 primu 3 gd 3% 50V 30'

(b)

.._, KH ·: K£du hitam KM : Kedu Merah KP : Kedu Putih

Figure 1. RAPD- PCR on 3% agarose gel with using primers 3 (a) KeduBlirik ( Kl3), Cemani ( C ) and Buras chicken ( AB ) on 3 % agarose gel; (b) KeduHitam ( KH ),KeduMerah ( KM ) and KeduPutih ( KP ).

(a) (b)

Figure 2. RAPD - PCR results3 % agarose gel with using primer4 (a) KeduPutih( KP ), KeduMerah (KM) and KeduBlirik (KB) on 3 % agarose gel, (b )Cemani (C) Buras chicken( AB ) and KeduMerah ( KM ) .

(a) (b)

Figure 3. RAPD - PCR results 3 % agarose gel with using primers 5 ( a)KeduHitam( KH ),KeduMerah(KM) and KeduB!irik (KB); (b) KeduPutih ( KP), KeduMerah(KM ) and KeduBlirik (KB) on 3 % agarose gel

140

2 Noy 2011 pri~'6 -.

""'3" 50\'"'

(a) (b)

Figure 4 . RAPD - PCR results3 % agarose gel with using primer 6 (a)KeduPutih (KP), KeduMerah (KM) and KeduBlirik (KB) on 3 % agarose gel, (b) KeduHitam (KH), Buras chicken (AB) and Cemani (C) .

The results of electrophoresis were analized, and then see if there is diversity among the

types of chicken DNA bands or in each groups of chicken. The diversity of DNA bands was

putted in the binary data tables, the presence of DNA bands was written with number one (I), the

disappearance of bands written with a zero (0). The diversity of DNA bands electrophoresis

results are presented in Table 2.

Table I. The binary data as a guide advent of DNA bands on agarose gel ( I :band; 0 :no band)

Primer 3 Primer 4 Primer 5 Primer 6 KHI 101 !!O.WO .. oJiooo 1110 KH2 101 001000 010001 !Ill AB2 011 Ollll{F ·101000

--. "-- 0 ~'' -Ill!

AB3 011 110110 111100 0011 AB4 Ill 011110 110100 Ill! ABS Ill 111110 110100 !Ill C2 101 .010110 . .OlHOO. UlQ C3 101 011110 OlllOO 0011 KPI 101 {)01010" i"Ooo.oo .. Ill! KP2 101 llll!O 010100 1111 KP3 101 01!110 .. •• or-ofo"O -.- ,,_._ __ ,___.,_,_ ·'--·-"'' ., llll KP4 !01 011110 0!0100 III I

. KM,1 -,~ .IQT .. . •oicl}io; ~:;~; .. ..·oJ!)o.ri . ··1111: KM2 101 111110 010111 1111 KM3 101 011110 0]()011 Ill! KM4 111 011110 010011 III I KB! 101 0010!6 Oll0l1 1111 KB2 101 001110 I 11000 !Ill KB3 !01 011110 110000 Ill!

141

The binary data obtained from the results of electrophoresis is converted into a ( adenine )

for the data binary one (written with I) and becomes g ( guanine ) for the binary numeral zero

(0). This is because the datas will be analyzed using the binary MEGA program, and the program

only recognizes MEGA constituent of DNA nucleotides ( A, T, G and C ) as well as 20 kinds of

amino acids that making up the protein. Replacement of data from the binary results of DNA

electrophoresis in letter 'a' ( replacement figure I ) and in letter 'g' ( substitute zeros ) are

presented in Table 2.

Table 2. Replacement of the binary to the nucleotide bases of data for analysis usmg the programMEGA ( I = a; 0 = g ).

. ' . ., ,y;I<H.J

KH2 1\BZ AB3

···AB4. AB5 •!t;:;2: C3

KpJ ...

KP2 ·. ~;3i··. KP4

!~l}' KM2

KM4

KB2 . '~~~):;!~

Primer 3

aga gaa gaa aaa aaa aga aga aga aga aga aga

:.3jg.r·· aga

<~~~ aaa

Primer4

ggaggg gaaa"8 aagaag gaaaag aaaaag gagaag gaaaag ggagag aaaaag gaaaa:g. gaaaag

· g,¥a~ag J

Primer 5 Primer 6

gaggga aaaa agaggg .. ,-.. aaaagg ggaa ~ga~ .. - -~ ::.;·'.:f";;~~~-~----

aagag~ aaaa gaaagg;,: .• . i •• ;_ ..•. ,.;s gaaagg ggaa

agggig> )iit!ii'T • . · gagagg aaaa . g~~w·;,,;-~~ '*~~'111'f·:; gagagg aaaa

·:-~~~~~~~~~~~~~!iri~~i}L:.:·._-aaaaag gagaaa aaaa ~R~,~g) _, __ -:_:_~r~:':£ :t~;;{i~~~~ttf~'l! -i;~lii¥!}%4~--: gaaaag gaggaa aaaa

;~li{~~:-c;·, .. /:_:::;~;_: <j~~i~:z~:Ilt~~~;~~:~l~--;~;,:_;:_~-:: "-' .-:~~~~l!~iM:;~ ~-:·~~~1 aga ggaaag aaaggg aaaa

,, _ , __ -;r{iga~;_:~'-:\:;:::-/:~::.:,:y fxa~gr~~;:~,::3~;~~·:?~~k:gi{\,~ ~:;sft~~i?:W"~W~;~>:_?; -::: _t

The variability of the electrophoresis results were analyzed by UPGMA method in the

MEGA program version 5. Genetic distance between individuals and groups were analyzed by

142

DNA band difference. Table 4 shows the diversity or differences among individuals and Table 3

shows the diversity among groups of chickens.

Table 3. Genetic distance orindividual differences based on differences in DNA band chicken results.

2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 I AB2 2 AB3 6 3 AB4 4 6 4 AB5 5 5 5 C2 7 7 5 6 6 C3 7 5 5 6 4 7 KBI 8 12 8 9 7 7 8 KB2 4 8 4 5 5 5 4 9 KB3 4 8 2 3 5 5 6 2 10 KHI 8 8 8 7 3 7 8 6 6 II KH2 9 13 7 8 8 8 3 5 5 7 12 KMI 7 II 5 6 6 6 3 5 3 7 4 13 KM2 9 9 5 4 6 6 5 7 5 7 6 2 14 KM3 7 11 5 6 6 6. 3 5 3 7 4 0 2 15 KM4 6 10 4 5 7 7 4 6 4 8 5 I 3 I 16 KPI 5 II 5 6 8 8 5 3 3 9 4 6 8 6 7 17 KP2 7 7 3 2 4 4 7 5 3 5 6 4 2 4 5 6 18 KP3 6 8 2 3 3 3 6 4 2 6 5 3 3 3 4 5 19 KP4 6 8 2 3 3 3 6 4 2 6 5 3 3 3 4 5 I 0

The analysis results of genetic diversity in this study are the genetic distances ranged

from 0 to 13. Genetic distance of zero ( 0 ), means that the chicken is no difference in the

banding pattern of the DNA of a total of 19 DNA bands were diverse. The chicken is a

KeduPutih 4to KeduPutih 3. Highest genetic distance at 13, is among the KeduHitam 2 to Buras

chicken 3. Genetic distance average is 5.30.

The lowest difference between groups of chicken in this study is4 and at most 9. The

smallest difference ( 4 ) in this study were between the KeduMerah toKeduB!irik and between

KeduPutih against KeduMerah and KeduB!irik. The biggest difference ( 9 ) is Kedu Black to

Buras chicken.

143

Table 4 . Genetic distance or difference between groups based on differences in DNA band chicken results RAPD - PCR using primers 3, 4, 5 and 6 using the program MEGA version 5.

2 3 4 5 6 A yam-buras

2 Ayam-cemani 6 3 Kedu-blirik 6 6 4 Kedu-hitam 9 7 6 5 Kedu-merah 7 6 4 6 6 Kedu-putih 5 5 4 6 4

Differences between chicken'sindividual DNA bands were analyzed using UPGMA

method in the MEGA program version 5 to get filogentik tree. Chicken phylogenetic tree in this

study are presented in Figure 5.

"''I KP3

""I KP4

KP2 ,,

I AS4

59 ABS

I 36

KM2

KM4

55 KM'

•• KM3

-= L,.[, ~~~ ,. 26

AB2

KP'

KB2

KB3

,---j KB'

K~

= 53 KH'

AB3

C3

3.5 3.0 0.5 0.0

Figure 5. Phylogenetic tree RAPD - PCR results using primers 3, 4, 5 and 6 in Buras chicken ( AB ), chicken KeduHitam ( KH ), chicken KeduMerah ( KM ), chicken KeduPutih( KP ), the chicken KeduBiirik ( KB ) and chicken Cemani ( C ) use the method of UPGMAin theprogram MEGA V.5.

Filogentik tree of the chicken was appears two main branches. Branch I is composed of3

Buras chicken and Cemani3, being the 2nd branch consists of I 7 other chickens. In the diagram

shows that Buras 3 and Cemani3 has the greatest difference against all other I 7 chickens, while

the KeduPutih 3 toKeduPutih4 and KeduMerah 1 have the least difference, compared to the hens

144

other. KeduMerah turns in the phylogenetic tree shows that to four chickens are clustered in a

separate sub-branch, which indicates that the diversity among individuals is small. This

possibility was appear because maybe KeduMerahsamples used in this study originated from a

hatchery, with the same stud, the same parent so diversity was little. Another samples were look

spreading at the sub-branch, it suggests a very high diversity. Therefore, no specific bands to

distinguish each type of chicken in this study, the results of RAPD - PCR using primers 3, 4, 5

and 6 can not be used as a genetic marker, but with RAPD - PCR results showed that the inter­

individual variability and between batches of chicken is still there, so it is still safe to be

cultivated.

CONCLUSION

1 . There are genetic diversity within populations and among populations, but can not be used as

a genetic marker

2 . Closest genetic distances areKeduBiirik to KeduKeduMerah and KeduPutih, as well as the

KeduMerah to KeduPutih.

3 . The most genetic distance is betweendomestic chicken and KeduHitam.

ACKNOWLEDGEMENTS

Thank youto thedean ofthe faculty ofveterinary medicineof UGMwhichhas provided the

opportunityforresearchanddevelopmentfundingso thatpartcan besolved bygoodresearch,

thanksare alsogiven tofriends in thebiochemistrylaboratory, to the committeewhichorganized an

international seminarso thatresearchcan bepublished.

145

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