LAPORAN PENELITIAN HIBAH PENELITIAN STRATEGIS NASIONAL Tahun Anggaran 2009
PENGEMBANGAN TEKNIK SELEKSI KUALITAS DAGING SAPI PO MENGGUNAKAN MARKA GEN KALPASTATIN
Oleh Dr. Sri Rahayu, M.Kes. Prof. Dr.Ir. Suyadi, M.S Agus Susilo, S.Pt., MP
Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Departemen Pendidikan Nasional, DIPA Universitas Brawijaya No. 0175.0/023-04.2/XV/2009 tanggal 31 Desember 2008, dan Berdasarkan SK Rektor Nomor : 160/SK/2009, tanggal 7 Mei 2009
UNIVERSITAS BRAWIJAYA NOPEMBER 2009
RINGKASAN Penelitian ini bertujuan untuk mendapatkan pola spesifik gen Kalpastatin (CAST) pada sapi PO yang berpotensi untuk dijadikan marka gen kandidat untuk seleksi. Dengan demikian, seleksi dapat dilakukan pada saat ternak masih berumur muda. Adapun tujuan jangka panjangnya adalah mendapatkan marka gen kandidat pengatur keempukan daging untuk seleksi ternak pada saat masih berumur muda, sehingga dapat meningkatkan efisiensi usaha peternakan secara signifikan. Penelitian dilaksanakan pada bulan Januari 2009 sampai dengan Oktober 2009. Pengamatan karakteristik fenotipik dan kualitas daging serta pengambilan darah sebagai sumber DNA dilakukan di RPH, Krian Sidoarjo. Analisis kualitas daging di laksanakan di Laboratorium Teknologi Hasil Ternak Fakultas Peternakan, Universitas Brawijaya, sedangkan analisis DNA dilakukan di Laboratorium Biomolekuler Fakultas MIPAJurusan Biologi, Universitas Brawijaya. Untuk mengembangkan marka gen yang cocok dilakukan seleksi sifat meat tenderness yang dilacak dengan primer gen CAST , dengan metode PCR-RFLP. Amplifikasi dilakukan dengan menggunakan primers Forward: 5’ATCCAGAAGACGGAAAGCCT-3’, sedangkan primer Reverse yang digunakan adalah : 5’-CTCACGATCCTCTTCTTTGG-3’Selanjutnya pemotongan hasil amplifikasi gen CAST sapi PO dilakukan melalui metode RFLP, dengan menggunakan enzim restriksi HaeIII. Dari penelitian yang dilakukan didapatkan bahwa nilai keempukan menunjukkan adanya variasi, yaitu kurang empuk (15 ekor), sedang (53 ekor) dan empuk (32 ekor). Hasil amplifikasi DNA menggunakan PCR pada ge CAST menghasilkan panjang fragmen dengan ukuran 600 bp. Hasil analisis polimorfisme didapatkan adanya tiga pola potongan pita DNA oleh enzim HaeIII, yaitu pola haplotip 1 dengan pola tidak terpotong oleh enzim HaeIII (600 bp), haplotip 2 dengan dua fragmen (600 dan 250 bp), dan haplotip 3 dengan tiga fragmen (600, 250, dan 350 bp).
SUMMARY Indonesia has the nations beef cattle, beef cattle both the local (Madura, Bali) or hybrid (Pernakan Ongole, Peranakan Limousin, etc.) with meat ternderness very varied. Until now the development effort has not been accompanied by the use of molecular technologies that can make an early selection of cows with a high meat tenderness that the efficient development efforts have not been met. Until now the selection of new meat quality appearance didasrakan top cut of meat after livestock. Have found a relationship between the marker Calpastatin (CAST) gene with meat tenderness (Barendse, 2002; Page et al., 2002, 2004; White et al., 2005). Calpastatin (CAST) inhibits activity and mcalpain associated with the regulation of the enzyme after cutting roteolisis. CAST increase in activity levels are closely related to meat tenderness. Schenkel et al (2008) found that polymorphisms in the CAST gene is closely associated with meat tenderness in cattle. The research Ciobanau et al (2004) shows a close relationship between the type of meat quality haplotip. This study aims to obtain a specific pattern of CAST genes in PO cattle, which potential to be a candidate gene markers for selection. Long-term goal is to get a candidate gene markers regulating meat tenderness cattle for selection at the age of the young, thus increasing farm efficiency significantly. Research conducted in January 2009 to October 2009. Observations phenotypic characteristics and meat quality as well as taking blood as a source of DNA made in slaugterhouse, Surabaya and Sidoarjo Krian. Meat quality analysis conducted at the Laboratory of the Faculty of Technology of Livestock Husbandry, Brawijaya University and DNA analysis conducted on Biomolecular Laboratory-FMIPA Biology Department , Brawijaya University. To get DNA fragmen, CAST gene were amplified by using DNA primer for Forward: 5’ATCCAGAAGACGGAAAGCCT-3 and Reverse primer: 5’CTCACGATCCTCTTCTTTGG-3’. The PCR products were digested by HaeIII the restriction endonuclease. Data obtained on the value of tenderness indicate a variation of beef tenderness on the PO cattle, which is less tender (15 samples), medium (53 samples) and soft (32 samples). Genetic influence on carcass quality of cattle to 48%, 61% of meat tenderness, fat thickness of 38%, LEA (Lean Eye Area) Longgisimus dorsi: 70% (Soeparno, 1998). The all PCR-product DNA was shown in size of 600 bp. Digestion by HaeIII restriction enzyme resulted 3 haplotypes. The first haplotype (haplotype I) was not cut by HaeIII enzyme, the second haplotype (haplotype II) producing two (2) fragments with molecular size of 600 and 250 bp. The third haplotype (haplotype III) producing three (3) fragments with molecular size of 600 , 250, and 350 bp. This patterns show there are polymorphisms for CAST gene of PO cattle. . The CAST gene variations cause by the random marriage, migration and selection in the population of PO cattle itself. Shackelford et al (1994) showed that approximately 65% of the variation in tenderness among cattle of all breeds was due to genetic effects. In an experiment with Angus bulls Palmer et al (1999) observed three genotypes and two alleles for CASTI and CAST5 loci and six genotypes AA, BB, CC, AB, AC and BC, for CAST10 locus. Single Nucleotide Polymorphisms (SNPs) in the calpastatin (CAST) gene was studied in Bos
Taurus (Jersey × Limousin, Angus and Hereford-cross cattle) by Morris et al (2006). Schenkel et al (2006) identified a SNP in the CAST gene (G to C substitution) in the crossbred commercial heifers, steers and bulls from the beef feedlots of the University of Guelph. They identified three genotypes (CC, CG and GG) and reported that the CAST’s SNP allele C was more frequent (63%) than Gallele. Kurly et al (2002) identified the polymorphism of Calpastatin gene with three restriction enzymes (Hinf I, MspI, RsaI) in Stambeek (Dutch Large white× Dutch Landrrace) pig breed.
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