Analisis genetik molekuler kuda (Equus caballus) lokal Pulau Bima berdasarkan sekuen gen cytochrome B

ASTASARI, Susthira , Yuriadi

2012 | Skripsi |

Kuda (Equus caballus) Bima merupakan kuda lokal asli Indonesia yang saat ini populasinya terancam punah. Berdasar data yang diambil dari Dinas Kesehatan Hewan NTB, populasi kuda Bima pada tahun 2008 sebanyak 65.988 ekor, selanjutnya pada tahun 2009 berjumlah 9.703 ekor. Sehubungan dengan hal tersebut perlu dilakukan tindakan konservasi secara in-situ dan ex-situ. Informasi mengenai karakter morfologi saja tidak cukup, sehingga perlu dilakukan kajian genetik untuk mengoptimalkan usaha konservasi. Tujuan penelitian ini adalah untuk menganalisa hubungan kekerabatan antar Kuda Bima terhadap Equus caballus berdasarkan gen penyandi protein cytochrome b. Sampel berasal dari tiga darah kuda yang berasal dari Desa Panda dan Desa Kalaki, Kecamatan Palibelo, Kabupaten Bima, Nusa Tenggara Barat. Amplifikasi segmen gen cytochrome b dengan metode PCR menggunakan hasil isolasi DNA untuk cetakan, primer CYTBECF dan CYTBECR. Reaksi PCR menghasilkan produk sepanjang 371 bp yang kemudian dilakukan sekuensing. Hasil sekuensing gen cytochrome b yang diperoleh selanjutnya dibandingkan dengan Equus caballus lain yang diambil dari Genbank. Hasil analisa dengan program MEGA versi 5.05 diperoleh 35 situs nukleotida yang berbeda dan 1 situs asam amino yang berbeda. Jarak genetik berdasarkan sekuen nukleotida gen cytochrome b yang dihitung menggunakan Kimura 2-parameter dihasilkan nilai sebesar 3,1%, dan nilai jarak genetik berdasarkan sekuen asam amino, yaitu 0,2%. Filogram menggunakan metode Neighbor Joining berdasar hasil urutan nukleotida dan asam amino gen cytochrome b dengan nilai bootstrap 1000 kali dapat digunakan untuk membandingkan kekerabatan kuda Bima dengan Equus caballus.

Bima horse is an indigenous horse of Indonesia which is consider an endangered species. Based on statistical data done by the local government of West Nusa Tenggara, its population was 65.998 in 2008, and drop into 9.703 in the year of 2009. Conservation efforts must be done to save this species, either by in-situ or ex-situ approaches. Morphological approaches are not sufficient, and a more basic approach need to be done, which is based on its genetic character. The aim of this study is to analyse the relationship between local Bima horses based on cytochrome b sequence of Equus caballus. Research samples as blood were collected from horses of 2 different areas in Western Nusa Tenggara, Panda and Kalaki villages of district Palibelo, Bima region. DNA obtained from the blood were used to run PCR reaction to amplify cytochrome b region by using CYTBECF and CYTBECR primers. A 371 bp fragment was produced and continued with sequence analysis. The resulting sequence was then compared with sequences database of Genbank. Sequence analysis with MEGA 5.05 detecting 35 different nucleotide sites and 1 different amino acid site between Bima horse and E. caballus. Genetic distance using Kimura 2-parameter based on nucleotide sequence of cytochrome b, the genetic distance among samples is 3.1%, and the genetic distance of 0.2% was obtained based on amino acids sequence. Phylogenetic analysis based on nucleotides and amino acids sequences by employing Neighbour-Joining method with 1000 bootstrap value revealed that cytochrome b could be used to study the relatedness of Bima horses, and it is shown that Bima horses are closely related to each other.

Kata Kunci : Equus caballus, DNA, gen cyt b, PCR, sekuen DNA