Carcass Traits Association with GH/AluI Gene Polymorphism in
Indonesian Aceh Cattle
Eka Meutia Sari¹,* , Ronny Rachman Noor2, Cece Sumantri2 & Endang Tri Margawati3
¹ Department of Animal Production, Faculty of Agriculture, Syiah Kuala University, Banda Aceh, *email: ekasalsa@yahoo.com
2Department of Animal Production and Technology, Bogor Agriculture University, Indonesia
3Research Center for Biotechnology (LIPI), Cibinong, Bogor, Indonesia
Abstract
This study was conducted in order to identify polymorphism of growth hormone gene in the exon five and to determine the association of GH/AluI polymorphism with carcass quality in Aceh cattle. A total of 42 DNA genome samples were extracted from two Aceh cattle population, i.e., Banda Aceh (12), Aceh Besar (30), and the PCR-RFLP was used to amplify 404 bp of GH gene. The result showed that the LL genotype was the only genotype found in Aceh cattle population, and the alele frequency of alel L is 1. This finding indicated that there was not evidence of polymorphism of GH/AluI in Aceh cattle, and there was not correlation of GH/AluI gene with carcass quality of Aceh cattle. It could be affected by small number of sampling size. However, this study suggests that GH gene could be possible used as genetic marker.
Keywords: Aceh cattle, GH gene, PCR-RFLP, Polymorphism
Introducton
The enhancement of beef cattle’s productvty n Indonesa wll be more approprate f t s done through a selecton whch s not only based on the phenotype, but also combned wth drect selecton on the level of DNA whch codes the phenotype n whch the qualty needs to be mproved. The bovne genome map made based on markers on genome DNA uses molecular technque such as RFLP, mcrosatellte, mnsatellte, PCR-RFLP, and PCR-SSCP make t possble to dentfy gene locus whch are responsble for trat varatons havng economc values.
2005). Casas et al. (2004) reported that QTL for growth trats, carcass composton and beef qualty was spread on chromosome 1, 2, 3, 16, 17, 19, 20, 21 and 26. GH gene has a great role n beef cattle’s performance (Breer. 1999), hence t s very nterestng to dentfy GH gene polymorphsm on Aceh cattle.
Materals and Methods
Ths research ncluded feld and laboratory actvtes. Feld actvty was con-ducted n Banda Aceh and Aceh Besar slaughterhouses. The DNA extracton and characterzaton of GH/Alu I gene dversty was conducted n Anmal Molecular Genetcs Laboratory, Faculty of Anmal Scence, Bogor Agrculture Unversty, whereas the examnaton of carcass and meat qualty was carred out n Rumnansa Besar Laboratory, Department of Anmal Producton and Technology. Ths actvty was done n October 2009 - November 2010.
Blood Sample of Aceh Cattle
Ths researchused Aceh cattle’s meat (muscle) n whch ths cattle s local ones orgnatng from Aceh provnce. Twelve (12) samples were taken n RPH Banda Aceh, and thrty (30) samples from RPH Antassalam, Aceh Besar (Table 1). The sample used was longisimus dorsi muscle on the 12th – 13th rb. The cattle slaughtered
were bred tradtonally, n whch durng daytme, they were herded, and put n stalls at nghts. The slaughter was done tradtonally.
Table 1 The number of meat sample used for GH/AluI gene analyss
Populaton Number
RPH Banda Aceh 12 RPH Aceh Besar 30
Total 42
Genome DNA Extraction (Meat)
DNA extracton was done from the meat. The extracton procedure followed phenol-chloroform method (Sambrook etal. 1989).
GH Gene Amplification
Table 2 Prmers GH/AluI (Gen Bank M57764.1, Gordone 2 Prmers GH/AluI (Gen Bank M57764.1, Gordon et al., 1983)
Locus Temperature
annealing
Product
PCR Sequences of Prmers
GH/AluI 59 ºC 404 bp F:5’-TAGGGGAGGGTGGAAAATGGA-3’ R:5’-GACACCTACTCAGACAATGCG-3’
Genotype Determination Using PCR-RFLP
The genotype determnaton of each ndvdual was done usng restriction fragment length polymorphism (RFLP) whch was vsualzed on agarose gel 1.5% wth the buffer of 0,5x TBE (trs borat EDTA), functoned on 100 V for 40 mnutes, and colored wth ethdum bromde on UV trans illuminator. AluI was utlzed as a cutter enzyme for the target gene fragment.
Data Analysis
Gene Frequency. The genotype frequency was the rato of the number of a certan genotype towards populaton number. Allele frequency was the rato of a certan allele towards the whole allele n a locus n a certan populaton. The frequency of each allele n each locus was counted based on Ne dan Kumar (2000) formula:
X = (2n + ∑nj) / (2N)
Results and Dscussons
GH Gene Amplification Results
The amplfcaton of growth hormone (GH) gene fragment done on Aceh cattle showed prmers forward on the poston of ntron 4 and prmers reverse on the poston of flakng regon 3 (Fgure 1). The GH gene fragment amplfcaton was conducted usng thermal cycler (Ependorf 5332) machne on the temperature of annealng 63 oC.
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The result of gene fragment amplfcaton vsualzed on agarose gel 1.5% s presented on Fgure 2. The length of GH gene fragment amplfcaton s 404 bp.
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Fgure 2. Vsualzaton of GH gene fragment amplfcaton result on agarose gel 1.5% (M: marker 100 bp, 1 -11: research sample)
The amplfcaton results conducted by Gordon et al. (1983), Yao(1983), Yao et al. (1996), Ge et al. (2003) and Zakzadeh(2003) and Zakzadeh et al. (2006) wth the same prmers showed that annealng prmers GH/AluI gene fragment on 59 ºC for 80 seconds, 65 ºC for 30 second, and 57 ºC for 60 seconds resulted n good PCR product. The annealng temperature used n ths research was 63 ºC for 45 second to obtan optmal PCR product so that t can be read clearly.
Identification of GH gene variants using PCR-RFLP
The determnaton of GH gene genotype n ths research was carred out usng PCR-RFLP wth AluI as the cutter enzyme. AluI enzyme recognzed AG|CT cuttng ste. Based on the sequence of GH gene DNA fragment beng amplfed, two AluI cuttng stes were obtaned; they are fragments wth the length of 87, 132, and 185 whch are knows as leucyne allele (L) (Pcture 3).
The cuttng usng AluI enzyme on AluI GH gene fragment as much as 404 bp only resulted n one knd of fragment: a fragment whch can be cut (two bars) known as LL genotype, whereas the fragment whch cannot be cut (one bar) known as VV genotype and combned fragment (three bars) knowns as LV genotype cannot be found n ths research (Fgure 3).
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Analysis Results on Aceh Cattle’ Carcass and Meat Quality
The value of parameter average of Aceh cattle carcass and meat qualty can be seen on Table 3. The pH value of the meat n ths research has the average of 5.46, whch shows the pH range of normal meat 5.4 – 5.8. The meat color s n category I (score 1 – 5) accordng to SNI (Indonesan Natonal Standard, 2008). The degree of tenderness 4 -5 s consdered moderate, not too tender and not too tough.
Table 3 The results of Aceh cattle’s meat and carcass qualty
Parameter n Average ± standard devaton fber and ts color s red. The qualty and meat structure greatly depend on types of meat and locaton. Marblng s also very nfluenced by the breedng system and food gven.
Conclusons
assocate wth the carcass qualty on Aceh cattle. Ths phenomenon s lkely due to the lmted number of samples and the exstence of natural selecton towards LV and VV genotype as the consequence of Aceh cattle adaptaton’s to the local env-ronment. Thus, a further research s stll necessary, by usng more samples and f dversty s found, sequencng needs to be done so that t results n more accurate research results.
References
Breer TA. 1999. Regulaton of proten and energy metabolsm by the somatotropc axs. Domest Anim Endocrinol 17:209-218.
Casas E et al. 2004. Identfcaton of quanttatve trat loc for growth and carcass composton n cattle.Anim Genet 35(1):2-6.
D Staso L et al. 2005. Polymorphsm of the GHR gene n cattle and relatonshpPolymorphsm of the GHR gene n cattle and relatonshp wth meat producton and qualty. Anim Genet 36:138-140.
Ge et al. 2003. Assocaton of a sngle nucleotde polymorphsms n growth hormone and growth hormone receptor genes wth blood serum nsuln-lke growth fac-tor I concentraton n Angus cattle. J Anim Sci 81:641-648.
Gordon DF et al. 1983. Nucleotde sequence of the bovne growth hormone chro-Nucleotde sequence of the bovne growth hormone chro-mosomal gene. Moll Cell Endocrinol. 33:81-95.
Sambrook J et al. 1989. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold
Sprng Harbor Laboratory Press, USA.
Yao J et al. 1996. Sequence varatons n the bovne growth hormone gene character-zed by sngle-strand conformaton polymorphsm (SSCP) analyss and ther assocaton wth mlk producton trats n Holstens. Genetics 144:1809-1816. Zakzadeh S et al. 2006. Analyss of bovne growth hormone gene polymorphsm n
three Iranan natve breeds and Holsten cattle by RFLP-PCR. Biotechnology
