Analisis 16s rDNA-mtDNA sebagai marka genetik variabilitas pertumbuhan ikan kerapu macan (Epinephelus fuscoguttatus) melalui metode PCR-RFLP

SUSANTO, Bambang, Dr.Drh. Widya Asmara, SU

2002 | Tesis | S2 Bioteknologi

Ikan kerapu macan (Epinephelus fuscoguztutus), tergolong Famili Serranidae, merupakan ikan ekonomis penting yang sedang dikembangkan sebagai salah satu komoditas unggulan untuk ekspor yang bernilai cukup tinggi. Produksi benih ikan tersebut telah berhasil dikembangkan dalam panti benih, tetapi masih ada kendala yaitu benih tumbuh berbeda ukuran dalam tiap periode pemeliharaan. Penelitian ini bertujuan untuk mengetahui marka genetik yang dapat dijadikan indikator variabilitas pertumbuhan pada benih ikan kerapu macan. Penelitian dilaksanakan di Laboratorium Bioteknologi pada Balai Besar Riset Perikanan Budidaya h u t - Bali. Pelaksanaan penelitian diawali dengan seleksi benih ikan berdasarkan ukuran panjang total dan berat yang berbeda. Benih ikan (umur 60 hari) ukuran besar memiliki panjang total dan berat masing-masing 63,27&2,62 mm dan 4,84&0,81 g; benih ikan ukuran sedang : 50,64*1,15 mm dan 2,35fl,22 g; benih ukuran kecil: 38,OWI ,39 mm dan 0,96kO,08 g. Ekstraksi mtDNA dilakukan dengan metode Ovenden (2000), amplifikasi fragmen 16s rDNA-mtDNA dengan PCR menggunakan primer Mt-I: 5'-CAT ATT AAA , CCC GAA TGA TAT TT-3' dan Mt-2: 5'-ATA ATA GGG TAT CTA ATC CTA GTT T-3'. Untuk mengetahui polimorfisme, 16s rDNA-mtDNA produk amplifikasi PCR dipotong dengan 4 enzim restriksi yaitu EcoRV, Hue-III, Hinf- I, dan Mbo I. Untuk mengetahui panjang fragmen DNA dan pola pita DNA, dilakukan elektroforesis agarose dan diamati dibawah UV trqsiluminator. Dilakukan interpretasi pemetaan pita DNA dan dianalisis dengan software program GENEPOP. Hasil amplifikasi PCR menunjukkan panjang fragmen 16s rDNA-mtDNA sekitar 1.948 bp. Pemotongan dengan EcoRV menghasilkan 2 pita, benih ikan besar 1233 bp dan 715 bp, benih sedang: 1242 bp dan 706 bp, benih kecil: 1241 bp dan 708 bp; dengan enzim Hinf 1 menghasilkan 2 pita, benih besar: 1518 bp dan 430 bp, benih sedang: 1521 bp dan 427 bp, benih kecil: 1521 bp dan 427 bp; enzim Mbo I menunjukkan 2 pita, benih ikan besar: 1324 bp dan 624 bp, benih sedang: 1320 bp dan 628 bp, benih kecil: 1331 bp dan 617 bp. Pemotongan dengan enzim restriksi Hae 111 menghasilkan pola pita yang berbeda untuk masing-masing ukuran benih. Benih ukuran besar menunjukkan 4 pita dengan panjang fragmen DNA: 1020 bp, 538 bp, 249 bp, 141 bp, benih sedang menghasilkan 4 pita : 991 bp, 556 bp, 269 bp, 131 bp. Sementara benih ikan berukuran kecil menghasilkan 3 pita : 1108 bp, 554 bp, 286 bp. Dari hasil interpretasi pola pita DNA dan jarak genetik antar populasi pada ikan berbeda pertumbuhan adalah antara ukuran besar dan ukuran sedang sebesar 0,007 , antara benih ukuran besar dengan benih berukuran kecil sebesar 0,249. Nilai F,, sebesar 0,96 1. Kesimpulan dari penelitian ini adalah pemotongan fragmen 16s rDNAmtDNA dengan HueIII dapat digunakan sebagai marka genetik variabilitas pertumbuhan dan variasi genetik benih ikan kerapu macan (Epinephelus firscoguttatus).

Groupers are an important commercial fish species in Indonesia and popular marine food fish of high market value in many parts of the world. The objective of this study was to find the genetic marker which can be used. as indicator variability of brown marbled grouper fries. The study was conducted at biotechnology laboratory, Research Institute for Mariculture on Bali. The initial study were selecting of fish fry based on the total length and body weight. The average total length of the large, medium, and small larvae were 63.27s.62 mm, 50.64k1.15 mm, and 38.09f1.39 mm respectively. The average of body weight of the large, medium, and small larvae were 4.84M.81 g, 2.35M.22 g, and 0.96M.08 g respectively. The fragment of 16srDNA-mtDNA were amplified with thermal PCR by using primer Mt-1: 5'-CAT ATT AAA CCC GAA TGA TAT TlT-3' and Mt-2: 5'-ATA ATA GGG TAT CTA ATC CTA GTT T-3'. The polymorphism fragment of product amplification of 16srDNA were digested with four restriction enzyme i.e. EcoRV, HueIII, Hinf I, and Mbo I. Agarose gel electrophoresis was used to assess the DNA fragment pattern and fragment length polymorphism. GENEPOP program was used to confirm genetic variation and genetic distance values between growth variability of grouper. Result of PCR amplification showed that molecule weight of 16srDNA fragment amplified was 1,948 bp. Digestion with EcoRV resulted in two fragment, on large, medium, and small, i.e. 1,233 and 7 15 bp, 1,242 and 706 bp, and 1,241 and 708 bp respectively. Similar result also showed on Hinf I, and Mbo I digestion, were two fragment length were obtained at large, medium, and small fry. Digestion with restriction enzyme Hinf I resulted 1,518 and 430 bp (large fry), 1,521 and 427 bp (medium fry), 1,521 and 427 bp (small fry) fragment. Restriction enzyme Mbo I give the result of 1,324 and 624 bp (large fry), 1,320 and 628 bp (medium fry), 1,33 1 and 617 bp (small fry). Digestion by using restriction enzyme HueIII that shown different fragment length pattern between fish fry. The large and medium fry were indicated 4 fragment length (1,020; 538; 249; and 141 bp), and (991; 556; 269; and 131 bp) respectively, whereas at small fish fry were indicated 3 fragment length i.e. 1,108; 554; and 286 bp. Genetic distance value inter pop with different growth rate were calculated 0.007 between large and medium size, while between large and small were 0.249. The standardized genetic variance (FSJ value about 0.96 1. The conclusion of this study were confirmed that HueIII restriction enzyme could digested of 16srDNA fragment and suggested to use as variability growth genetic marker of brown marbled grouper fry.

Kata Kunci : Bioteknologi,Marka Genetik,Ikan Kerapu Macan, 16srDNA, variability growth genetic marker, E. fuscoguttutus, PCRRFLdP