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Raja Ampat Berdasarkan Gen Identifikasi Synaptula (Echinodermata : Holothuroidea) Raja Ampat Berdasarkan Gen COI Robitoh Desi Kurniasari 1 , Aris Soewondo 2 , Abdul Hamid Toha 3 1), 2) Jurusan Biologi FMIPA Universitas Brawijaya Malang 3) Jurusan Biologi FMIPA Universitas Papua Email korespondensi : Robitoh D.K ; [email protected] ABSTRAK Identifikasi spesies yang terkoleksi di perairan Raja Ampat perlu dilakukan untuk mendapatkan data biodiversitas perairan Raja Ampat. Synaptula merupakan salah satu timun laut dan merupakan organisme salah satu organisme yang cryptic species. Penelitian ini bertujuan untuk mengidentifikasi Synaptula yang dikoleksi dari perairan Raja Ampat menggunakan metode molekuler dengan marker gen COI. Alasan memilih Synaptula untuk diidentifikasi dikarenakan spesiemen tersebut masih teridentifikasi sampai tingkat genus. Metode identifikasi secara molekuler yang digunakan adalah DNA barcoding, dengan tahapan, isolasi DNA, PCR, dan sekuensing. Hasil penelitian yang telah dilakukan menunjukkan bahwa Synaptula dari Raja Ampat (UNP 101) memiliki kemiripan yang tinggi dengan spesies Plakobranchus ocellatus dari Sulawesi dan Philipina, namun Synaptula UNP 101 dari Raja Ampat dengan Synaptula reciprocans jarak genetik sebesar 76.8% dan nilai similaritas sebesar 23.6% sehingga dapat diartikan bahwa memiliki perbedaan yang besar. Perbedaan hasil identifikasi kemungkinan disebabkan oleh kontaminasi DNA template Synaptula oleh DNA template Plakobranchus UNP 67A dikarenakan proses identifikasi dilakakukan dalam waktu yang bersamaan. Kata Kunci : gen COI,identifikasi, Synaptula . ABSTRACT Identification of species Raja Ampat required in order to obtain marine biodiversity in Raja Ampat. Synaptula is one of the organism that inhabit in Raja Ampat and belonging to sea cucumber . Sea cucumber is an organism that difficult to distinguish through morphological characteristic.. This study aimed to identify Synaptula from Raja Ampat based on mitochondrial gene, cytochrome c oxidase subunit 1 (COI). The results of the study that Synaptula of Raja Ampat (UNP 101) is Plakobranchus ocellatus , the result is different with identification through morphological. Synaptula of Raja Ampat (UNP 101) has a high similarity with Plakobranchus ocellatus species from Sulawesi and the Philippines. Genetic distance Synaptula of Raja Ampat (UNP 101) with Synaptula reciprocans is 76.8%, while similarity values is 23.6%. The difference in the results of species identification may be caused by contamination DNA Plakobranchus UNP 67A to DNA Synaptula UNP 101, contamination is likely to occur during the identification process which simultaneity. Keywords: COI gene, identification, Synaptula . PENDAHULUAN Identifikasi terhadap Synaptula perlu dilakukan karena diperlukan pendataan Raja Ampat merupakan gugusan pulau- terhadap keragaman spesies di Indonesia. pulau kecil di pulau Papua. Lokasi kepulauan Identifikasi terhadap Synaptula memerlukan Raja Ampat merupakan ekosistem koral metode identikasi molekuler, karena Synaptula Indonesia yang paling asli dan paling kaya. merupakan cryptic spesies . Metode Potensi sumberdaya terumbu karang yang identifikasi molekuler yang digunakan adalah dimiliki kabupapten Raja Ampat, merupakan DNA barcoding dengan penanda gen COI bagian dari “segitiga karang dunia” ( coral (mtDNA). Pada hewan, penggunaan mtDNA truangle ) yang terdiri dari Indonesia, Filipina, dalam identifikasi spesies didasari oleh Papua New Guinea, Jepang, dan Australia. analisis biogeografi dan sistematik sering tidak Raja Ampat memiliki ekositem yang paling sejalan dengan identifikasi morfologi. Salah kaya diantara pulau-pulau penyusun segitiga satu penyebabnya adalah karakter morfologi karang [1]. Synaptula merupakan salah satu seringkali memperlihatkan fenomena species spesies timun laut yang ditemukan di Raja cryptic. Sedangkan mtDNA hewan merupakan Ampat. genom sitoplasmik yang diwariskan secara Jurnal Biotropika | Vol. 2 No. 5 | 2014 265 uniparental dan tidak mengalami rekombinasi buffer (1X), dNTPs (masing-masing dNTP 0,3 sehingga species sibling bisa dipastikan mM (1X)), MgCl 2 (2 mM 1X) dan stabiliser) mempunyai mtDNA dengan nilai kesamaan sebanyak 10 µl, 1 µl masing-masing primer yang tinggi. Salah satu ruas mtDNA yang (10 µM), dan 1 µl DNA template. Program banyak digunakan sebagai barcode yaitu PCR : hot start 95°C selama 3 menit, cytochrome oxidase 1 (CO1) yang denaturation 95°C selama 30 detik, annealing dipopulerkan oleh Hebert et al . (2003). Gen 50°C selama 30 detik, extension 72°C selama cytochrome oxidase 1 (CO1) adalah segmen 1 menit(30 siklus), dan post extension 72°C dari DNA mitokondria yang terdiri atas 648bp selama 1 menit. Sampel hasil PCR disimpan [2]. pada suhu -20 0C. Hasil PCR kemudian Synaptula merupakan salah satu spesies dilakukan Elektroforesis gel agarose untuk yang tergolong kedalam ordo apodida. melihat DNA amplikon yang didapatkan dan Karakter morfologi apodida adalah tubuhnya panjang base pair. yang silindris, sistem pernafasannya DNA amplikon hasil PCR kemudian di mengerucut ke atas bagian tubuh, tidak sekuensing untuk mengetahui susunan basa memiliki papilla pada bagian anal, memiliki pada organisme makroinvertebrata Synaptula spikula atau ossicles yang sering termasuk Raja Ampat. Sekuen yang didapatkan dari kedalam golongan anchors yang berfungsi sekuensing kemudian dianalisis menggunakan untuk attachment kedasar substrat dan software bioedit dan disejajarkan dengan data membantu pergerakannya sebagai alternative base di NCBI. Hasil pensejajaran kemudian alat gerak menggantikan tidak adanya struktur dipilih sekuan yang memiliki kesamaan diatas podia. Keberadaan anchors ini merupakan 85% dengan sekuen sampel Synaptula. sekuen kekhasan yang dimiliki famili synaptidae dan yang dipilih kemudian dialigment dan perbedaan struktur anchors dapat digunakan disamakan panjang sekuen dan dilakukan sebagai salah satu karakter morfologi yang perhitungan jarak genetic menggunakan membedakan antar genus pada family software Mega 5.1. Berdasarkan jarak genetic synaptidae [3]. Synaptula yang ditemukan di yang telah didapatkan dihitung nilai perairan Raja Ampat perlu diidentifikasi similaritasnya dengan persamaan (1-jarak dengan metode DNA barcoding dengan genetik) X 100% kemudian hasilnya penanda gen COI sehingga didapatkan jenis diterjemahkan kedalam pohon filogenetik taksanya dan data yang didapatkan dapat untuk mengetahui hubungan kekerabatan antar digunakan untuk kepentingan manajemen spesies berdasarkan gen COI menggunakan konservasi. maximum likehood (ML) dengan metode estimasi jarak p-distance (boostrap 1000) pada METODE PENELITIAN software MEGA 5.1 . Penelitian bertempat di Laboratorium HASIL DAN PEMBAHASAN Biologi Molekuler Jurusan Biologi Fakultas MIPA Universitas Brawijaya. Sampel DNA isolasi dari jaringan organisme makroinvertebrata Synaptula (UNP 101) Synaptula menggunakan Gsync DNA diambil pada 4 Juni 2013 di aljuy bay, Waigeo ExtractionKit (Geneaid). DNA hasil isolasi dan disimpan dalam botol berisi etanol 95%. dilihat kualitasnya dengan uji kualitatif Ekstraksi DNA Synaptula dilakukan elektroforesis gel agarose . menggunakan Gsync DNA Extraction Kit (Geneaid). Kemudian Elektroforesis Gel Agrose Hasil Isolasi DNA untuk melihat hasil ekstraksi DNA. DNA yang telah diisolasi diamplifikasi sekuen gen CO1 dengan primer universal gen CO1 LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’ dan HCO2198, 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’[4]. Racikan untuk PCR terdiri dari ddH2O 12µl (diganti dengan Gambar 1: hasil uji kualitatif isolasi DNA larutan BSA), pcr mix kapa Taq Extra Synaptula (yang ditunjuk garis merah merupakan HotStart ReadyMix PCR kit (1,25 U per 50µl pita DNA yang Synaptula ) reaksi DNA polimerase, KAPA Taq Extra Jurnal Biotropika | Vol. 2 No. 5 | 2014 266 DNA specimen berhasil diisolasi namun Hipotesis tersebut didasarkan pada menunjukkan smear yang kemungkinan hasil alignment dari sampel J1,J2,K1,K2,dan disebabkan DNA yang terisilasi belum murni UNP 67A sebagai berikut ; masih terdapat kontaminan seperti protein dan RNA. DNA yang didapatkan kemudian dilakukan amplifikasi gen COI dengan metode PCR menggunakan primer LCO-1490 dan HCO-2198 . Gen COI digunakan sebagai marker genetic dikarenakan gen COI memiliki Gambar 3. Hasil aligment sekuen hasil sekuensing tingkat evolusi yang tinggi sehingga dapat Synaptula dan UNP 67A* (beda basa* digunakan untuk membedakan meski dalam nukleotida ditunjuk oleh tanda bintang) kekerabatan yang dekat. Gen COI digunakan sebagai marker genetic dikarenakan gen COI Dari hasil alignment menunjukkan memiliki tingkat evolusi yang tinggisehingga bahwa hanya terdapat 2 basa yang berbeda dapat digunakan untuk membedakan meski (ditandai dengan tanda bintang ) antara dalam kekerabatan yang dekat. Synaptula UNP 101 (J1,J2,K1, dan K2) Berdasarkan hasil uji kualitatif gel terhadap UNP 67A ( Plakobranchus ocellatus ) agarose 0.8% diketahui bahwa sekuen gen dan didapatkan hasil BLAST yang memiliki COI dapat teramplifikasi dengan panjang kemiripan yang tinggi dengan Plakobranchus. amplikon ±750 bp namun pita DNA hasil Hal ini berbeda dengan identifikasi amplifikasi tipis (ditunjuk oleh kotak merah ) morfologi yang merujuk pada spesies hal ini kemungkinan disebabkan oleh DNA Synaptula. Hal ini kemungkinan disebabkan Synaptula yang terisolasi belum murni oleh kontaminasi DNA template Synaptula sehingga sekuen yang teramplifikasi saat PCR sedikit. DNA Synaptula diulang sebanyak 4 dengan DNA tempalate Plakobranchus tube dalam proses PCR. dikarenakan proses pengerjaan proses identifikasi yang bersamaan.
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