Hubungan kekerabatan sapi lokal berdasarkan markah genetik cytochrome b (Cyt b) dengan analisis PCR-RFLP

CAHYADI, Muhammad, Tety Hartatik, S.Pt., Ph.D

2009 | Tesis | S2 Bioteknologi

Penelitian ini bertujuan untuk mengetahui hubungan kekerabatan sapi lokal berdasarkan gen cytochrome b (cyt b) dengan analisis PCR-RFLP. Materi yang digunakan adalah jaringan telinga dan sampel darah sapi Bali, Madura, PO dan sapi lokal di Kabupaten Pacitan. Sampel jaringan telinga dikoleksi dari Rumah Potong Hewan (RPH), sedangkan sampel darah diambil dari sapi di Daerah Istimewa Yogyakarta (DIY), Kabupaten Pacitan, Pulau Madura, Pulau Bali, Kalimantan dan Nusa Tenggara Barat (NTB). Total DNA diisolasi dari sampel menggunakan DNA KIT high pure PCR template preparation (ROCHE) dan metode standar Sambrook et al. (1989). Amplifikasi fragmen gen cyt b menggunakan forward primer L14735 (5’-AAA AAC CAC CGT TGT TAT TCA ACT A-3’) dan reverse primer H15149 (5’-GCC CCT CAG AAT GAT ATT TGT CCT CA-3’). Produk PCR yang dihasilkan didigesti dengan enzim restriksi HinfI, TaqI, PstI dan EcoRI. Hasil digesti produk PCR dielektrofresis pada agarose 2%. Hasil analisis PCR-RFLP menunjukkan bahwa sapi lokal Indonesia memiliki tiga haplotipe, yaitu haplotipe A, haplotipe B, dan haplotipe C. Penentuan haplotipe didasarkan pada perbedaan pola restriksi produk PCR oleh enzim HinfI dan TaqI. Enzim PstI dan EcoRI tidak memotong produk PCR. Perbedaan haplotipe ini menandakan bahwa sapi lokal Indonesia memiliki karakteristik genetik yang beragam. Phylogenetic tree dihasilkan dari analisis sekuen fragmen gen cyt b menggunakan software MEGA4. Hasil analisis menunjukkan bahwa sapi Bali, sapi Madura, sapi lokal Pacitan (Pc9, Pc13, PO1P dan PO2P) berkerabat dengan Banteng/Bos javanicus (AY689188), sedangkan sapi PO dari DIY (PO5 dan PO22) dan sapi Lokal Pacitan (Pc11) berkerabat dengan sapi dari bangsa Bos indicus (AF492350). Ovis aries (NC_001941) digunakan sebagai out group.

The research was conducted to evaluate genetic relationship of domestic cattle based on PCR-RFLP cytochrome b (cyt b) analysis. Both ear tissues and bloods from Bali, Madura, Ongole-grade (PO), Local Pacitan cattle were used as samples. Ear tissues were collected from slaughterhouses, while blood samples were collected from cattle in DIY Regency, Pacitan Regency, Madura Island, Bali Island, Kalimantan and NTB. The DNA was isolated by using DNA KIT high pure PCR template preparation (ROCHE) and standard method from Sambrook et al. (1989). The DNA genome was used as a template to amplified cyt b gene by using forward primer L14735 (5’-AAA AAC CAC CGT TGT TAT TCA ACT A-3’) and reverse primer H15149 (5’-GCC CCT CAG AAT GAT ATT TGT CCT CA-3’). The RFLP analysis was performed by digest the PCR products with HinfI, TaqI, PstI and EcoRI restriction enzymes. Digested PCR product was electrophoreted on 2% agarose gels. The PCR-RFLP analysis showed that Indonesian domestic cattle had three haplotype, there were haplotype A, haplotype B, and haplotype C. The haplotypes were determined by differentiation of restriction patterns of PCR products with HinfI and TaqI restriction enzymes. Both PstI and EcoRI were not restricted the PCR products. The differentiation of haplotypes were indicated that Indonesian domestic cattle had genetic diversity. The sequence of cyt b genes fragment were analyzed by MEGA4 computer’s software to showed phylogenetic tree. The results showed that Bali cattle, Madura cattle and Local Pacitan cattle (Pc9, Pc13, PO1P and PO2P) related to Banteng/ Bos javanicus (AY689188), while Ongole-Grade cattle from DIY (PO5 and PO22) and Lokally Pacitan cattle (Pc11) related to Bos indicus (AF492350) cattle breed. Ovis aries (NC_001941) was used as out group.

Kata Kunci : Kekerabatan,Sapi lokal,Cyt b,PCR,RFLP, Relationship, Domestic Cattle