Appendix 1. Tools on reseach. Microwave. Waterbath Incubator. Centrifuge Kubota Research. Horizontal Mini gel Kit. Geneaid Purification Kit
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2 26 Appendix 1. Tools on reseach Waterbath Incubator PTC 100 Research Master Microwave Cycler MJ Centrifuge Kubota 3740 Horizontal Mini gel Kit Geneaid Purification Kit UV illuminator Microcentrifuge
3 27 Appendix 1. Continued Heater and stirer Vacuum desicator Digital weighing machine Eppendorf microtube Micropipette Vortex mixer
4 28 Appendix 2. Laboratory analysis steps DNA Extraction PCR Amplification +primer L15267 & H15526 (for Cytochrome b) +primer RAG2F2 & RAG2R6 (for RAG2) + Using PTC 100 Master Cycler MJ Research Electroforesis Using Horizontal Mini gel kit + 2% Agarose gel DNA estimation Using UV iluminator Result DNA Purification DNA Sequencing Using Macrogen Service DNA sequence data ATGGCAAGCCTACGCA AAACGCACCCCCTCAT TAAAATCGCACCCCCT CATTAAAATCGCACCC Fluorogram Text data
5 Appendix 3. Fluorogram of sequencing result using Automatic Sequencer 3730xl machine. A. Forward primer 29
6 Appendix 3. Continued B. Reverse Primer 30
7 31 Appendix 4. R script using APE packages library(ape) cytb.botia < read.dna("botia CYTOCHROME COMPLET.txt", "sequential") cytb.botia summary(cytb.botia) dist.cytb.botia < dist.dna(cytb.botia) #!!! Kimura's distance dist.cytb.botia hist(dist.cytb.botia) hist(dist.cytb.botia, breaks = 40) summary(dist.cytb.botia) tree.nj.cytb.botia < nj(dist.cytb.botia) #!!! Neighbor joining tree.nj.cytb.botia plot(tree.nj.cytb.botia) add.scale.bar(x = 0.05, length = 0.01) title("neighbor joining tree for Botia cytochrome b") rag2.botia < read.dna("botia RAG2 COMPLET.txt", "sequential") rag2.botia summary(rag2.botia) dist.rag2.botia < dist.dna(rag2.botia) #!!! Kimura's distance dist.rag2.botia hist(dist.rag2.botia) summary(dist.rag2.botia) tree.nj.rag2.botia < nj(dist.rag2.botia) #!!! Neighbor joining tree.nj.rag2.botia plot(tree.nj.rag2.botia) add.scale.bar(x = 0.005, length = 0.001) title("neighbor joining tree for Botia RAG2")
8 Appendix 5. Table of genetic distance inter individual of botia based on mtdna Cytochrome b Sampel BBH1 BBH2 BBH4 BBH5 BBH6 BTB1 BTB2 BTB3 BTB4 BTB5 BBH2 0, BBH4 0, , BBH5 0, , , BBH6 0, , , , BTB1 0, , , , , BTB2 0, , , , , , BTB3 0, , , , , , , BTB4 0, , , , , , , , BTB5 0, , , , , , , , , BM1 0, , , , , , , , , , BM2 0, , , , , , , , , , BM3 0, , , , , , , , , , BM4 0, , , , , , , , , , BM5 0, , , , , , , , , , BKP1 0, , , , , , , , , , BKP2 0, , , , , , , , , , BKP3 0, , , , , , , , BKP4 0, , , , , , , , BKP6 0, , , , , , , , , , Information : BBH : Botia Batanghari BTB : Botia Tulang Bawang BM : Botia Musi BKP : Botia Kapuas 32
9 Appendix 5. continued Sampel BM1 BM2 BM3 BM4 BM5 BKP1 BKP2 BKP3 BKP4 BM2 0, BM3 0, , BM4 0, , , BM5 0, , , , BKP1 0, , , , , BKP2 0, , , , , , BKP3 0, , , , , , , BKP4 0, , , , , , , , BKP6 0, , , , , , , , , Information : BBH : Botia Batanghari BTB : Botia Tulang Bawang BM : Botia Musi BKP : Botia Kapuas 33
10 Appendix 6. Table of genetic distance inter individual of Botia based on nucleus DNA RAG2 Sampel BBH1 BBH2 BBH4 BBH5 BBH6 BTB1 BTB2 BTB3 BTB4 BTB5 BBH2 0, BBH4 0, , BBH5 0, , , BBH6 0, , , , BTB1 0, , , , , BTB2 0, , , , , , BTB3 0, , , , , , , BTB4 0, , , , , , , , BTB5 0, , , , , , , , , BM1 0, , , , , , , , , , BM2 0, , , , , , , , , , BM3 0, , , , , , , , , , BM4 0, , , , , , , , , , BM5 0, , , , , , , , , , BKP1 0, , , , , , , , , , BKP2 0, , , , , , , , , , BKP3 0, , , , , , , , , , BKP4 0, , , , , , , , , , BKP6 0, , , , , , , , , , Information : BBH : Botia Batanghari BTB : Botia Tulang Bawang BM : Botia Musi BKP : Botia Kapuas 34
11 Appendix 6. Continued Sampel BM1 BM2 BM3 BM4 BM5 BKP1 BKP2 BKP3 BKP4 BM2 0, BM3 0, , BM4 0, , , BM5 0, , , , BKP1 0, , , , , BKP2 0, , , , , , BKP3 0, , , , , , , BKP4 0, , , , , , , , BKP6 0, , , , , , , , , Information : BBH : Botia Batanghari BTB : Botia Tulang Bawang BM : Botia Musi BKP : Botia Kapuas 35
12 CURRICULUM VITAE Author was born in Pasuruan, East Java on October 23 th 1984 whose father H.Machfudz, SH. and mother Hj. Siti Rofi ah. Author is the youngest son from 2 sisters and 1 brother. His elementary school graduated on year 1996 in MIN Darul Ulum Jombang, on 1999 author graduated his junior high school in MTsN Plandi Jombang. On 2002, author graduated his senior high school in SMUN 2 Jombang, and on that year he pass the selection test entering IPB through USMI. Author was accepted in Department of Living Aquatic Resource Management, Faculty of Fisheries and Marine Science. During student, author is active as official of some student organizations such as Koperasi Mahasiswa (KOPMA) Institut Pertanian Bogor period , Badan Eksekutif Mahasiswa (BEM) Fakultas Perikanan dan Ilmu Kelautan period , Himpunan Mahasiswa Manajemen Sumberdaya Perairan (HIMASPER) period , also become chief of Majelis Ta lim Al Marjan Fakultas Perikanan dan Ilmu Kelautan period Author also became assistant in subject Introduction of Computer and Algorithmic on 2004/2005. On 2007 author do his profession as webmaster in some institutional website such as Agroedutourism FAPET IPB, Taman Nasional Sembilang, and El diina. In order to complete his study, author did the research on joint project of Institut de Recherche pour le Développement (IRD), France with Badan Riset Kelautan dan Perikanan Departemen Kelautan dan Perikanan (BRKP DKP), Indonesia, with dissertation title Genetic Differentiation among Populations of Chromobotia Macracanthus Bleeker from Sumatera and Kalimantan Based On Sequencing Gene of mtdna Cytochrome B and Nucleus DNA RAG2.
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