The 6th ISTAP lnternational Semi4ar on Tropical Animal Production

uru

"Integrated Approach in Developing Sustainable Tropical.Animal Production" ,i' 4',

PROCEEDINGS PART II

October 20-22,2015 Yogyakarta Indonesia

ISBN:

97 8-979

-1215-26-8

Published by: Faculty of Animal Science, Universitas Gadjah Mada Yogyakarta,Indonesia,2015

T

Editor-in-Chief Cuk Tri Noviandi (Universitas Gadjah Mada, Indonesia)

Editorial Board Subur Priyono Sasmito Budhi ZaenaI Bachruddin

Ristianto Utomo Widodo Soeparno Yuny Erwanto

Adiarto Ismaya Tety Hartatik Wihandoyo Endang Baliarti Krishna Agung Santosa Sudi Nurtini Budi Guntoro Nanung Danar Dono Zuprizal Keshav L. Maharjan Henning Otte Hansen Yukinori Yoshimura Allen Young Yanin Opatpatanakit

(Universitas Gadj ah Mada, Indonesia) (Universitas Gadjah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadjah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadjah Mada, Indonesia) (Universitas Gadjah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Universitas Gadj ah Mada, Indonesia) (Hiroshima University, Japan) (University of Copenhagen, Denmark) (Hiroshima University, Japan) (Utah State University, USA) (Maej o University, Thailand)

Editorial Staff Rima Amalia EW, Prisilia Putri S, Miftahush S Haq, Septi Mulatmi, Aditya Alqamal, Riyan Nugroho A, Pradiptya AH, Satyaguna R, Zefany aAG, B agas Pamungkas

International seminar on Tropical Animal Production Production integrated Approach in Developing sustainable Tropical Animal October 20-22, 2015, Yogyakarta, Indonesia

The

6,h

Identification of GhlAlu-I Gene Poly_morphisms in Indonesian Simeulue Buffalo Etmy Marianar' Eka Meutia Sarir, Mohd. Agus Nashri Abdullahr, Mohd' Yunusr' Eryk Andreas2 rFaculty of Agriculture, Dept. of Animal Science, Syiah Kuala University, Banda Aceh 23111 2Facuhv olAnimal Science, Bogor Agricultural Universiry Jl. Agatis, DarmagaCampus, Bogor 16680 Corresponding E-mail: ekasari865@yahoo'com gene polymorphism in ABSTRACT: The purpose of this study was to identis the GwAluI data on the

this is the first published Indonesian Simeulue Uuffato. To the besi of our knowledge The 178 DNA samples buffalo polymorphism of growth hormcne (GH) gene in Simeuluqbuffalo. Teupah Selatan (71), Teupah Barat (59)' were collected from three districts in Siireulue Island, of ttre cHlatul gene at432 bp located on and Salang (48). Result shows thatagene fragment of PCR (polymerase chain reaction) exon 3 wefe successfully amplified by using th"e techniques lengih polymorphism)' Based on that results and genotyped by pCn-nff.i'(restriction fr"agment g.n.r. Alibuffaloes tested had LL genotype for showed no polymorphisms were detected in tiese locus GHlAluI.

Keywords:SimeulueBuffalo,GrowthHormoneGene,Polymorphism

INTRODUCTION Indonesia. They are also regarded as Buffalo is one of the importance domestic animals in for- its,products, attention has been focused the excellent meat producer. To increasing demand local buffalo is a source of germplasm that can on the genetic imprtvement of these ,p..i".r. The public welfare, to create employment be used in order to increase food availability, to improve genetically adapted to specific environmental and to generate foreign exchange. Animals tiat are be deveroped using low cost, supporting the conditions would be Tnore proJuctive because it can effective in achieving the objectives of food diversity of food, agriculture and culture, as well as security (FAO, 2000). (GH) is an anabolic hormone synthesizt^1-uld secreted ol ".tlll 2006)' GH has an important role in the somatotropin in anterior pituitary (Ayuk and sheppard, -^.i-- li-irlc anl

"l]:l:

8r:";;ffi;;e

;;ffiH;";;;;tdeveropme"t,sl?ytllt::"'.'.ti:t1t':'l"ll:*l'H:'.1*ll?tp|l:tffii et al',zoos)' The studv of GH gene Mspl (ikers

#il;t""#r

*.,*"tism

,iooe;ThidarMyint

--1r1 ^ /o--+^*^ -t ^l 10O6'Srrtarno o..n reported in Hereford and composite cattle !Sr11a3o er ",r.,,rr::::.,::: Pesisir cattle (Jakarta et al.' 2007), Aceh 1998), ongole Grade (Po) cattle (Sutarno et a\.,2005), -t a1.,2013t ^l ,n1?\ jli,"Ji|il?")i |,'.,"|ou), rndonesia rocar buffaio (Andreas et ar., z0t0; Sumantri.i et rL^ ^i* of nf this thi in Simeulue buffalo has never done' rhe aim ;XffiJ"":: /rtLI\ ot ^ano nt polymorphism of growth hormone^ (GH) gene research was conducted in order to identify the AluI loci in Indonesian Simeulue buffalo'

;ffirffi.,"**

Jri;ffi;;r;;i.."ir.^*.ter

MATERIALAND METHODS DNA SamPle 410

'. : ' International Seminar

''

on Tropical Animal Production :.:ated Approach in Developing Sustainable Tropical Animal Production -. :er 20-22,2015, Yogyakarta, Indonesia

DNA samples obtained from blood buffalo. The blood samples were used as a source of originating from three different regions, namely 59 samples from "-: --l.lh as 178 DNA samples *::h Barat, 71 samples from Teupah Selatan, and 48 samples from Salang.

r Pnmers

!''r-ritrn e

gene segments of GH followed Balogh et al- (2009), with -,,.r,c primer 5'-CGGACCGTGTCTA|GAGAAGCTGAAG-3', and reverse primer 5'ength was 432bp. :-IaTTGAGCAGCGCGTCGTCA-3'. The amplified product length :r\ { E\traction

to amplify

DNA was extracted from blood buffalo. Extraction procedure followed the phenol chloroform -. :,::,rJ (sambrook and Russell, 2001) was modified with the following procedure:

rmple preparation

$

The blood in the alcohol were as much as 200 pl. Sample was inserted to a 1.5 ml tube' - : _ rol *,as eliminated from the sample by adding distilled water until 1000 pl, and left in room rpm for : --r jrstlrro for 20 minutes. Then it was precipitated by centrifugation at a speed of 8,000 '' -:. -:.Ites. I rr; tein degradation

-

The samples were cleared from alcohol and added by 200 pL 1x STE (sodium tris EDTA), were incubated --- sodium dosesil sulfate l0o/o, and20 pl proteinase K (5 mglml). The mixture -::,rght at 55 'C temperature while shaken gently.

rtenic material degradation \fier incubated, samples were added by 400 ptl phenol solution, 400 pL chloroform/isoamyl . , . htrl (24:l), and 40 pt- Strl NaCl. Then, the mixture was shaken at room temperature for one "

Ix\

\ precipitation

, :i

phase Samples were centrifuged at a speed of 5,000 rpm for 10 minutes to separate the water phenol phase. Water phase was transferred in a new tube with the volume measured. DNA

* . ,:;ules

x volume of 5M NaCl. oC - -.::.1 rhe mixture was incubated at a temperature of -20 over night. Subsequent DNA was -.::pitated by centrifugation at a speed of 12,000 rpm for 10 minutes. Obtained DNA precipitate : :: rrashed by 70% aliohol, and then precipitated again. Precipitated DNA clean from alcohol -::r -.re d by adding 100 pl TE (Tris EDTA). DNA samples were stored at -20 oC and ready for use. deposited by adding a 2x volume of alcohol absolute and 0.1

trnplification of GH Gene Amplification of GH fragment was done by using PCR (polymerase chain reaction) methods. ; ;,rents used for amplification of both target fragment were a 2 pL sample DNA, each primer -: rmol, 200 prM dNTPs mixture, 1 mM MgCl, and 0.5 units of DreamTaqrM DNA Polymerase ,- j tr buffer (Fermentas) in total solution 25 ytL.Amplification in vitro within Gene Amp@ PCR : ..:em 9,700 (Applied Bio systemsrM) done with the condition of pra-denaturation at 94'C for 5 *--1utes, 35 cycles consisting of denaturation at 94"C for 45 seconds, annealing primers at62'C '.: -15 seconds and extension of new DNA at 72'C for I minute, and the final extension at 72oC rtlenotyping by using RFLP Method

::

Determination of genotypes of each individual was done by using restriction fragment length x TBE buffer (tris borate ^l.morphism (RFLP), follow by visualize d on2o/o agarose gel with 0.5

47r

t\ Production The 6th International Seminar on Tropical Animal Animal Production Integrated Approach in Developing Sustainable Tropical October 20 -22, 2015, Yogyakarta' Indonesia

EDTA) at 100

on UV v for 40 minutes. Gel was stained by ethidium bromide, and visualized AluI'

transiluminator. Cuttin

g

gene was enzymethat is used for both sides of the Iarget

Genotype and Allele FrequencY to total population' Allele frequency Genotype frequency represents the ratio of a genotype population. Mathematical model genotype is a ratio of an allele to the overall allele at a locus inlhe as follows: and allele frequency (Nei and Kumar, 2000) is represented r,.

-'11 *rcrr";

s $

.t f\t{

a rr n.. I x.

t\ ltllltryr iral1hllsf =ntull'"r''.tlrq,)r,11 il ts"r!{\*' * rrrxnber x*4rlf 6f il ,tfll]n?f

=

-

tFl3l

-

rs

srTli

nltrlr ii('r:l*fit!'

RESULTS AND DISCUSSION

Amplification of Buffalo GH Gene fragm"lt Ylt carried on Gene Amp@::.Tjtt:::1"t"1(3iti:' .-rr gene Amplification of GH o-.-* *-"o;;"^ -'\rrrPr'rv4!rv'vr raltzed on 1.50 -t^ ^*-liGo'l nanc Biosystemsrg with temperature of 62"C' rhe amplifitd et-n:19T:lt:::::tll::"" 4tlor 4th bp of *ui +zz bp' incruding 55 hn gH,s'i' ;#:JJ:';:i +tfiintron, und qg Up of Stn exon (Baloghet al''2009)' -

iil;;;;';il;;"d*;;q1

.ion,

,-:*:y

Method Identification of GH Gene by Using PCR-RFLP study was done by PC* *l-t-,it^tl:1^Tt:: Determination of GH gene genotypes in this sesmenl r-."nafci. e"t.a on DNA sequences of GH11senes< amplified Al.,r #;;;;;';;e t ir ?A{ hn bp . and 265 ^n , 5l , 96, ^^A fragments of length 20 ;H H"J';;J." ;;, AluI cutting, which produced t\ ^-l Qrrraonr Sumanrr. and (2010) eas et al ailele (L). This results also supported by Andr 1 lf\fll\ tlr: -n so th' (Lucv et, at',1ee3), 1758 c to G atposition (I)qlooh ,r --^r:-^ ^11^1^ /\/\ ot nl lii.'e,r' 20, t4i,.Td26? 2y,\1"I* il.ii?l*T:,1::lY]"!Tt""il"?u,lo,, populatiorg.r ,r'o*.0 ttrat ttre GHlAluI locus the three buffalo ---r^ /I]:^-,*^ 1\ LL genotype was found in a total sample (Figure 1)'

fi'iil;r;;il; f}|,[ilj:;'*;; ";:;:;"il^;#

""'Ii"*,i";f."*

i,,ilffii#ilffi;;t:;;ffi was monomorphic. The

KS?s KS33 KS45

KG53

SCIfl lrP

265

3flO bp

btP

?oo bp 9S trp

140 bp

Agarose Gel' M: DNA Ladder 100 bp' Figure 1. Visualization of the GHlAluI locus on 2oh Buffalo SamPles GenotYPe LL

472

tL

'

-

I

Production The 6'h International Seminar on Tropical Animal Animal Production Integrated Approach in Developing Sustainable Tropical October 20-22, 2015, Yogyakarta, Indonesia

simeulue Buffalo Genetic Diversity of GHlAluI Genes within Indonesian the allele frequency. Allele frequency Level of diversity within populations can be drawn from in one population. Information on genetic is a ratio of one allele relative to the overall allele found by the value of heterozygosity (Nei diversity of a population using multiple loci can be described and Kumar, 2000).

Genetic diversity based on molecular marker by the value of one genotype GHlAluI loci in buffalo were very low. This was indicated the fixation process' Low diversify in frequency and allele which had a value of 1, which marks population buffalo can be caused by a limited number of males in the

use of GH/AluI onlv resulted in LL Based on this research, it can ..to".)|nl:::]n. a marker' This phenomenon is likely genotype and monomorphic, so that it cannot be used as of natural selection towards LV and VV due to the limited number of samples and the existence to the local environment' Thus' a genofype as the consequence of 3imeulue buffalo adaption's and if diversity is found' sequencing further research is still necessary, by using -or" ,u-ples needs to be done so that the results are more accurate'

ACKNOWLEDGMENTS The work was supported by

DIKTI Republic of Indonesia through the Hibah Bersaing 2014

project.

REFERENCES

A' 2010' Identification of GH/ Andreas E, Sumantri C, Nuraini H, Farajjalah A, and Anggraeni J' Indonesian Trop' Anim' AluI and GHfu AluI Genes fotymorphism in Indoneiian Buffalo. Agric, 35:215-221. ancl growth factor regulation of Akers RM. 2006. Major advances associated with hormone 89:1222-12234' mammary g.owth and lactation in dairy cows. J. Dairy. Sci. Postgrad' Med' J' 82:24-30' its disorders' and Ayuk J and Sheppard MC. 2006. Growth hormone A, Fesus L, Delavaud c' chilliard Balogh o, Kovacs K, Kulcsar M, Gaspardy A, Febel H, Zsolnai Interrelationship of growth hormone AluI X Gilbert Ro and Huszenicza dy..zooq. metabolites in the beginning polymorphism and hyperketonemia with plasma hormones and of lactation in dairy cows. Livestock Sci' 123:180-186' Shere BD' (Ed')' Food Agriculture FAO. 2000. world watch List for Domestic Animal Diversity, Organrzatton of the United Nations, Rome, Italy' Evaluasi Keragaman Genetik Gen Jakaria, Duryadi D, Noor RR, Tappa B and Marlojo H.2007 ' Penciri PCR-RFLP' Hormon perltumbuhan Sapi Pesisir Sumatera Barat Menggunakan Media Peternakan, 30: 1 - 1 0. DE and collier R'I' 1993' Variants Lucy MC, Hauser SD, Eppard PJ, Krivi GG, clark JH, Bauman milk of somatotropin in cattle: gene frequencies in major dairy breeds and associated production. Domest. Anim. Endocrinol ' 10:325-333 ' University Press, New Nei M and Kumar S. 2000. Molecular Evolution and Phylogenetic. Oxford York.

Manual, 3rd ed' Cold Spring Sambrook J and Russell D. 2001. Molecular Cloning: A Laboratory Harbor Laboratory Press, United State of America' 413

The 6,hInternational seminar on Tropical Animal Production Integrated Approach in Developing Sustainable Tropical Animal Production October 20 -22, 20I 5, Yogyakarta, Indonesia

of Single Sari EM, Noor RR, Sumantri C, Yunus M, Han JL and Muladno. 2013. Identification Nucleotide polymorphism on Growth Hormone Gene in Aceh Cattle. Media Peternakan, 2r-23. growth Sutarno, Lymbery AJ, Thompson RCA, and Cummins JM. 1996. Association between cattle' of beef growth hormone genotypes ani estimated breeding value for preweaning proceeding of ih. 13th International Congress on Animal Reproduction. Sydney June 30Jl;'ly 4,p: 19-26 Biology. Sutarno. 199g. Candidate gene marker for production traits in beef cattle. In: Veterinary Perth: Murdoch UniversitY. gen hormon pertumbuhan Sutarno, Junaidi A, Tappa n. zoos. Polymorfisme MSPI pada lokus 2 Biodiversitas 6:77 -81' harian. sapi pO do, p"rlLruhnya terhadap capaian berat badan of Growth Hormone Sumantii C, AnggraenlA, Suri EM, Andreas E. 2013. Genetic Polymorphism SAADC. Genes in Indonesian Local Buffalo. Proceeding The 4th International Conference LanzhouUniversity. China 2l -31 July, p : 216-219' administration of ThidarMyint H, YoshidaH, Ito T, Inoe ff an-a Kuwayama H. 2008' Combined calves' preweaning Holstein ghielin and GHRH synergistically stimulates GH release in Domest. Anim. Endocrinol . 34:118-123'

474