Phylogenetic relationships of stress resistant fish in Harapan Rainforest Jambi based on DNA barcode

Abstract

DNA barcode as an effective tool for identification and reveal phylogenetic relationships in fish. The purpose of this study was to analysis DNA sequence, genetic distance and reveal phylogenetic relationships of stress resistance fish (blackfish) in Harapan Rainforest Jambi base on DNA barcode. The research was conducted from April to August 2016 in Harapan Rainforest Jambi, Integrated Laboratory, Jambi University and Biotechnology Laboratory of the Primate Study Centre-IPB University. DNA extraction was done on five blackfish species from Harapan Rainforest Jambi, namely Nandus nebulosus, Pristolepis grootii, Trichopodus leerii, Channa striata and Channa micropeltes. As a comparison, we used stress intolerance fish (whitefish) from Harapan Rainforest Jambi, i.e., Balantiocheilos melanopterus and Hemibagrus nemurus from the gene-bank NCBI with acession number KT001040,1. DNA Extraction was performed according to Quick-Star Tissue Protocol from Qiagen. COI gene amplification with modification at denaturation and anneling temperatures. Visualization DNA band using a horizontal electrophoresis machine from Bio Rad. Sequencing DNA send to 1st Base Malaysia. DNA sequence used Biodit and MEGA X software. The alignment of the DNA bands in MEGA X produces DNA sequence along 588 bp, where 350 bp conserve and 238 bp variable sites. The composition of the base nucleotides were (T/U) =29%, C=28.6%, A=25%, and G=17.3%. The closest genetic distance was between Channa striata and Channa micropeltes (0.190)  and the farthest was found on  Nandus nebulosus and  Hemibagrus nemurus (0.303). The phylogeny tree shows that the blackfishes are separated from whitefishes. The group of blackfish is divided into Channidae group (Channa striata, Channa micropeltes) and non-Channidae group (Nandus nebulosus, Trichopodus leerii, Pristolepis grootii).

Abstrak

DNA barcode sebagai alat efektif  identifikasi dan mengungkap hubungan filogenetik pada ikan. Penelitian ini bertujuan untuk menganalisis runutan DNA, jarak genetik dan hubungan filogenetik ikan tahan cekaman (blackfish) di Hutan Harapan Jambi berdasarkan DNA barcode. Penelitian  dilakukan pada April-Agustus 2016 di Hutan Harapan Jambi, Laboratorium Terpadu Universitas Jambi dan Laboratorium Pusat Studi Satwa Primata IPB Bogor. Ektraksi DNA dilakukan pada lima spesies ikan tahan cekaman dari Hutan Harapan  Jambi yaitu kerapu rawa (Nandus nebulosus), sepatung (Pristolepis grootii), sepat mutiara (Trichopodus leerii), gabus (Channa striata), dan toman (Channa micropeltes). Sebagai kelompok pembanding  digunakan ikan yang tidak tahan cekaman (whitefish) dari Hutan Harapan Jambi yaitu ridiangus (Balantiocheilos melanopterus), dan dari gen-bank NCBI yaitu baung (Hemibagrus nemurus) nomor akses KT001040,1. Ektraksi  DNA berdasarkan Quick-Star Protocol  jaringan dari Qiagen. Amplifikasi gen COI dengan modifikasi pada suhu penguraian DNA dan penempelan primer. Visualisasi pita DNA menggunakan mesin elektroforesis horizontal dari Bio Rad. Sequencing DNA dikirim ke 1^st Base Malaysia. Analisis DNA menggunakan perangkat lunak Bioedit dan MEGA X. Hasil pensejajaran pita DNA pada MEGA X menghasilkan runutan DNA sepanjang 588 bp, terdiri atas conserve 350 bp dan variable 238 bp. Komposisi basa nukleotida (T/U) =29%, C=28,6%, A=25%, dan G=17,3%. Jarak genetik terdekat pada Channa striata dan Channa micropeltes 0,190 dan  terjauh pada Nandus nebulosus dan Hemibagrus nemurus 0,303. Berdasarkan pohon filogeni menunjukkan bahwa kelompok  blackfish (ingroup)  terpisah dengan kelompok whitefish (pembanding), pada blackfish  terbagi atas Channidae (Channa striata, Channa micropeltes) dan non Channidae (Nandus nebulosus, Trichopodus leerii, Pristolepis grootii).